MEMCAL 

LUIBMAl^Y 


IN  HEMORIAM 
O.W.  JONES,  SR. 


BOOKS 


JOSEPH  McFARLAND,  M.  D. 


Pathogenic  Bacteria  and  Protozoa 
Octavo  of  878  pages,  illustrated.     Cloth, 

$3.50  net.  Seventh  Edition 


Pathology 

Octavo  of  856  pages,  with  437  illustra- 
tions.    Cloth,  $5.00  net.       Second  Edition 


Biology:  General  and  Medical 

i2mo  of    440  pages,   with   160    illustra- 
tions.   Cloth,  $1-75  net. 


A  TEXT-BOOK 


UPON  THE 


PATHOGENIC  BACTERIA 
AND  PROTOZOA 


FOR 


STUDENTS  OF  MEDICINE  AND 
PHYSICIANS 


BY 

JOSEPH  MCFARLAND,  M.D. 

Professor  of  Pathology  and  Bacteriology  in  the  Medico-Chirurgical  College, 

Philadelphia;  Professor  of  Pathology  in  the  Woman's  Medical  College 

of  Pennsylvania ;  Pathologist  to  the  Philadelphia  General  Hospital 

and  to  the  Medico-Chirurgical  Hospital,  Philadelphia;  Director 

of  the  Laboratories  of  the  Henry  Phipps  Institute  ;  Fellow 

of  the   College   of   Physicians  .of  Philadelphia,  etc. 


TOtb  293  flllustrations,  a  number  of  tbem  in  Colors 


Seventh  Edition,  Thoroughly  Revised 


PHILADELPHIA  AND  LONDON 

W. B.    SAUNDERS    COMPANY 
1912 


Copyright,  1896.  by  W.  B.  Saunders.  Reprinted  September,  1896.  Re- 
vised, reprinted,  and  recopyrighted  August,  1898.  Reprinted  November, 
1898.  Revised,  reprinted,  and  recopyrighted  August,  1900.  Reprinted 
June,  1901.  Revised,  entirely  reset,  reprinted,  and  recopyrighted  May, 
1903.  Reprinted  August,  1904.  Revised,  reprinted,  and  recopyrighted 
May,  1906.  Reprinted  August,  1907.  and  May,  1908.  Revised,  reprinted, 
and  recopyrighted  August,  1909.  Revised,  reprinted,  and  recopyrighted 
September,  1912. 


Copyright,   1912,  by  W.  B.  SAUNDERS  COMPANY. 


PRINTED    IN    AMERICA 


PRESS    OF 

W.    B.    SAUNDERS    COMPANY 
PHILADELPHIA 


TO 

MY  HONORED  AND  BELOVED  GRANDFATHER 

jflfcr,  5acob  (Brim 

WHOSE    PARENTAL   LOVE  AND  LIBERALITY  ENABLED  ME  TO  PURSUE 
MY   MEDICAL   EDUCATION 

THIS  BOOK   IS  AFFECTIONATELY  DEDICATED 


PREFACE  TO  THE  SEVENTH  EDITION. 


To  his  scientific  friends,  whose  continued  appreciation 
and  patronage  have  made  necessary  the  preparation  of  this 
seventh  edition  of  the  PATHOGENIC  BACTERIA,  the  author 
desires  to  extend  his  sincere  thanks. 

In  so  far  as  his  pages  have  been  found  a  useful  and  reli- 
able guide,  he  is  elated;  in  so  far  as  they  may  have  failed, 
he  feels  humiliated,  but  'is  stimulated  to  renewed  and  more 
earnest  endeavors  on  their  behalf. 

The  flight  of  time  has  brought  with  it  many  changes,  but 
perhaps  in  no  department  of  learning  have  they  come  in 
greater  number  or  with  more  startling  rapidity  than  in 
Microbiology. 

When,  some  eighteen  years  ago,  the  author  was  appointed 
to  give  the  first  systematic  course  of  lectures  upon  Bacteri- 
ology, in  the  Medical  Department  of  the  University  of  Penn- 
sylvania, there  were  few  text-books  suitable  for  the  use  of 
students,  and  the  preparation  of  the  "Pathogenic  Bacteria" 
seemed  to  be  a  justified,  though  a  doubtful  venture.  To-day 
our  shelves  groan  beneath  the  weight  of  many  excellent  vol- 
umes. 

When  the  " Pathogenic  Bacteria"  appeared,  all  of  the 
existing  books  were  general  in  character.  The  title  adopted 
by  the  author  seemed  to  be  of  doubtful  expediency,  lest  it 
should  limit  the  success  of  the  work  by  contracting  the 
sphere  of  its  usefulness.  But  it  was  fortunate  in  meeting 
with  a  cordial  reception,  and,  in  spite  of  its  title,  soon 
came  to  be  looked  upon  and  used  as  a  general  text-book. 
All  of  the  early  revisions  were  directed  toward  increasing 
its  general  usefulness  and  making  it  serviceable  in  all  the 
fields  in  which  Bacteriology  was  taught  or  practised. 

But  now,  times  have  changed,  and  it  can  no  longer  be  said 
that  anything  short  of  a  many  volume  encyclopedia  can  be 
regarded  as  an  adequate  "general"  work  upon  Bacteriology. 
There  are  now  excellent  books  devoted  to  microbiology;  to 

7 


8  Preface 

the  systematic  classification  and  identification  of  bacteria; 
to  the  laboratory  methods  used  in  studying  them;  to  the 
chemistry  and  toxicology  of  their  metabolic  products ;  to  the 
problems  of  infection  and  immunity;  to  the  individual  the- 
ories of  immunity;  to  the  blood-serum  therapy;  to  bacterio- 
vaccination  and  the  opsonic  index;  to  complement-fixation; 
to  the  bacteriology  of  water;  to  the  bacteriology  of  foods; 
to  the  bacteriology  of  the  dairy ;  to  the  bacteriology  of  sew- 
age and  the  methods  of  its  disposal ;  to  the  relation  of  bacte- 
riology to  agriculture ;  to  the  relation  of  bacteriology  to  the 
public  health;  to  veterinary  bacteriology,  and  so  on  and  on, 
almost  without  limit.  A  dozen  great  international  journals 
in  English,  German,  and  French  are  devoted  to  the  subject, 
and  weigh  down  our  shelves  with  hundreds  of  ponderous 
volumes  of  innumerable  monographs  and  experimental  re- 
searches, and  one  becomes  bewildered  in  his  efforts  to 
"keep  up"  with  the  ever-expanding  information. 

In  the  meantime  the  "Pathogenic  Bacteria,"  diverted 
from  one  after  another  of  the  fields  it  had  pre-empted,  but 
for  which  it  was  not  definitely  intended,  found  and  held  its 
own  place  as  a  medical  book. 

As  it  became  more  and  more  clear  that  the  original  inten- 
tion of  the  author  was  to  be  realized  and  the  destiny  of  his 
book  was  to  be  purely  medical,  it  became  equally  clear  that 
the  present  revision  must  meet  the  requirements  of  that  field 
as  completely  and  as  perfectly  as  possible. 

In  the  past  the  "Pathogenic  Bacteria"  has  been  devoted 
to  the  consideration  of  bacteria  only  When  the  author  was 
criticized  because  it  had  nothing  to  say  about  the  higher 
fungi  and  the  Protozoa,  he  pointed  out  that  the  title  de- 
clared the  contents  of  the  work,  and  a  change  would  be 
inconsistent  with  the  original  purpose. 

There  was  always  the  feeling  that  the  development  of 
Protozoology  would  soon  make  it  necessary  for  the  student 
to  have  a  text-book  upon  the  Pathogenic  Protozoa,  and  that 
it  would  then  become  necessary  to  divorce  the  two  subjects 
again.  As,  however,  knowledge  of  the  protozoa  engaged  in 
human  pathology  has  not  so  expanded  as  to  make  this  either 
necessary  or  desirable,  and  as  the  future  purpose  of  the 
"Pathogenic  Bacteria"  is  to  meet  the  needs  of  students  of 
human  medicine  and  pathology,  it  has  become  both  desir- 
able and  practicable  to  change  the  original  plan,  depart  from 
the  unwholesome  consistency,  and,  without  any  important 


Preface  9 

change  in  the  title,  offer  to  old  friends  and  future  patrons  a 
work  that  shall  endeavor  to  meet  all  modern  requirements : 

By  describing  all  the  pathogenic  micro-organisms  of 
importance  in  human  medicine,  whether  they  be  bac- 
teria or  protozoa; 

By  teaching  the  laboratory  technic  with  reference  to 
the  needs  of  medical  students  and  practitioners; 

By  bringing  each  micro-organism  under  consideration 
into  a  historic,  geographic,  biologic,  and  pathologic 
setting ; 

By  dwelling  upon  the  anatomic  and  physiologic  disturb- 
ances referable  to  the  various  micro-organisms; 

By  describing  the  lesions  occasioned  by  the  different 
micro-organisms;  and, 

By  explaining  such  methods  of  diagnosis  and  treatment 
as  grow  out  of  the  knowledge  of  microbiology  in  gen- 
eral and  of  the  micro-organisms  in  particular. 

JOSEPH  McFARLAND. 
PHILADELPHIA,  September,  1912. 


CONTENTS. 


PART  L— GENERAL. 


PAGE 

HISTORICAL,  INTRODUCTION 17 

CHAPTER  I. 
STRUCTURE  AND  CLASSIFICATION  OF  THE  MICRO-ORGANISMS 29 

CHAPTER  II. 
BIOLOGY  OF  MICRO-ORGANISMS 58 

CHAPTER  III. 
INFECTION 78 

CHAPTER  IV. 
IMMUNITY 105 

CHAPTER  V. 
METHODS  OF  OBSERVING  MICRO-ORGANISMS 172 

CHAPTER  VI. 
STERILIZATION  AND  DISINFECTION 201 

CHAPTER  VII. 
CULTURE-MEDIA  AND  THE  CULTIVATION  OF  MICRO-ORGANISMS...   224 

CHAPTER  VIII. 
CULTURES  AND  THEIR  STUDY 242 

CHAPTER  IX. 

THE  CULTIVATION  OF  ANAEROBIC  MICRO-ORGANISMS 260 

11 


12  Contents 

CHAPTER  X.  PAGE 

EXPERIMENTATION  UPON  ANIMALS 268 

CHAPTER  XI. 
THE  DETERMINATION  OF  BACTERIA 278 

CHAPTER  XII. 
BACTERIOLOGY  OF  THE  AIR 283 

CHAPTER  XIII. 
BACTERIOLOGY  OF  WATER 287 

CHAPTER  XIV. 
BACTERIOLOGY  OF  THE  SOIL 294 

CHAPTER  XV. 
BACTERIOLOGY  OF  FOODS 296 

CHAPTER  XVI. 
DETERMINATION  OF  THE  THERMAL  DEATH-POINT  OF  BACTERIA.  .  300 

CHAPTER  XVII. 

DETERMINATION  OF  THE  VALUE  OF  ANTISEPTICS,  GERMICIDES, 

AND  DISINFECTANTS 302 

CHAPTER  XVIII. 

THE   PHAGOCYTIC  POWER  OF  THE  BLOOD  AND  THE  OPSONIC 

INDEX 307 

CHAPTER  XIX. 
THE  WASSERMANN  REACTION  FOR  THE  DIAGNOSIS  OF  SYPHILIS.  . .   318 


PART  IL— THE  INFECTIOUS  DISEASES  AND 
THE  SPECIFIC  MICRO-ORGANISMS. 


CHAPTER  I. 
SUPPURATION 339 


Contents  13 


CHAPTER  II.  PAGE 

MALIGNANT  EDEMA 374 

CHAPTER  III. 
TETANUS 385 

CHAPTER  IV. 
ANTHRAX 4°° 

CHAPTER  V. 
HYDROPHOBIA,  LYSSA,  OR  RABIES 412 

CHAPTER  VI. 
CEREBROSPINAL  MENINGITIS 423 

CHAPTER  VII. 
GONORRHEA 43 1 

CHAPTER  VIII. 
CATARRHAL  INFLAMMATION 439 

CHAPTER  IX. 
CHANCROID 442 

CHAPTER  X. 
ACUTE  CONTAGIOUS  CONJUNCTIVITIS 46 

CHAPTER  XI. 
DIPHTHERIA 45 1 

CHAPTER  XII. 
VINCENT'S  ANGINA 478 

CHAPTER  XIII. 
THRUSH 484 

CHAPTER  XIV. 
WHOOPING-COUGH..  .  488 


CHAPTER  XV. 
PNEUMONIA 492 


14  Contents 

CHAPTER  XVI.  PAGE 

INFLUENZA 514 

CHAPTER  XVII. 
MALTA  OR  MEDITERRANEAN  FEVER 520 

CHAPTER  XVIII. 
MALARIA 524 

CHAPTER  XIX. 
RELAPSING  FEVER 546 

CHAPTER  XX. 
SLEEPING  SICKNESS 554 

CHAPTER  XXI. 
KALA-AZAR  (BLACK  FEVER) 566 

CHAPTER  XXII. 

YELLOW  FEVER 576 

CHAPTER  XXIII. 
PLAGUE 581 

CHAPTER  XXIV. 
ASIATIC  CHOLERA 604 

CHAPTER  XXV. 
TYPHOID  FEVER 632 

CHAPTER  XXVI. 
DYSENTERY 687 

CHAPTER  XXVII. 
TUBERCULOSIS '. 710 

CHAPTER  XXVIII. 
LEPROSY 762 

CHAPTER  XXIX. 

GLANDERS 775 


Contents  15 

CHAPTER  XXX.  PAGE 

RHINOSCLEROMA 785 

CHAPTER  XXXI. 

SYPHILIS 787 

CHAPTER  XXXII. 
FRAMBESIA  TROPICA  (YAWS) 800 

CHAPTER  XXXIII. 

ACTINOMYCOSIS 803 

CHAPTER  XXXIV. 
MYCETOMA,  OR  MADURA-FOOT 812 

CHAPTER  XXXV. 
BLASTOMYCOSIS 818 

CHAPTER  XXXVI. 

RINGWORM 824 

CHAPTER  XXXVII. 

FAVUS..  .  828 


BIBLIOGRAPHIC  INDEX 833 

INDEX.  .  849 


PART   I.    GENERAL 


HISTORICAL  INTRODUCTION. 

BIOLOGY,  chemistry,  medicine,  and  surgery,  in  their  evolu- 
tion, contributed  to  a  new  branch  of  knowledge,  Bacteri- 
ology, whose  subsequent  development  has  become  of  in- 
estimable importance  to  each.  Indeed,  bacteriology  illus- 
trates the  old  adage,  "  The  child  is  father  of  the  man," 
for  while  it  is  in  part  the  offspring  of  the  medicine  of  the 
past,  it  has  established  itself  as  the  dictator  of  the  medicine 
of  the  present  and  future,  especially  so  far  as  concerns  the 
infectious  diseases. 

THE  EVOLUTION  OF  BACTERIOLOGY. 

I.  BIOLOGIC  CONTRIBUTIONS  j  THE  DOCTRINE  OF  SPONTANEOUS 
GENERATION. 

Among  the  early  Greeks  we  find  that  Anaximander 
(43d  Olympiad,  610  B.  C.)  of  Miletus  held  the  theory  that 
animals  were  formed  from  moisture.  Empedocles  of 
Agrigentum  (450  B.  C.)  attributed  to  spontaneous  genera- 
tion all  the  living  beings  which  he  found  peopling  the 
earth.  Aristotle  (384  B.  C.)  is  not  so  general  in  his  view 
of  the  subject,  but  asserts  that  "sometimes  animals  are 
formed  in  putrefying  soil,  sometimes  in  plants,  and  some- 
times in  the  fluids  of  other  animals." 

Three  centuries  later,  in  his  disquisition  upon  the  Pytha- 
gorean philosophy,  we  find  Ovid  defending  the  same  doc- 
trine of  spontaneous  generation,  while  in  the  Georgics  Virgil 
gives  directions  for  the  artificial  production  of  bees. 

The  doctrine  of  spontaneous  generation  of  life  was  not 
only  current  among  the  ancients,  but  we  find  it  persisting 
through  the  Middle  Ages,  and  descending  to  our  own  genera- 
tion. In  1542,  in  his  treatise  called  "  De  Subtilitate,"  we 

2  I7 


1 8  introduction 

find  Cardan  asserting  that  water  engenders  fishes,  and  that 
many  animals  spring  from  fermentation.  Van  Helmont 
gives  special  instructions  for  the  artificial  production  of 
mice,  and  Kircher  in  his  "  Mundus  Subterraneus  "  (chapter 
"De  Panspermia  Rerum")  describes  and  actually  figures 
certain  animals  which  were  produced  under  his  own  eyes 
by  the  transforming  influence  of  water  on  fragments  of 
stems  from  different  plants.* 

About  1671,  Francesco  Redi  seems  to  have  been  the  first 
to  doubt  that  the  maggots  familiar  in  putrid  meat  arose  de 
novo:  "Watching  meat  in  its  passage  from  freshness  to  de- 
cay, prior  to  the  appearance  of  maggots,  he  invariably  ob- 
served flies  buzzing  around  the  meat  and  frequently  alight- 
ing on  it.  The  maggots,  he  thought,  might  be  the  half- 
developed  progeny  of  these  flies.  Placing  fresh  meat  in  a  jar 
covered  with  paper,  he  found  that  although  the  meat  putre- 
fied in  the  ordinary  way,  it  never  bred  maggots,  while  meat 
in  open  jars  soon  swarmed  with  them.  For  the  paper  he 
substituted  fine  wire  gauze,  through  which  the  odor  of  the 
meat  could  rise.  Over  it  the  flies  buzzed,  and  on  it  they 
laid  their  eggs,  but  the  meshes  being  too  small  to  permit 
the  eggs  to  fall  through,  no  maggots  generated  in  the  meat ; 
they  were,  on  the  contrary,  hatched  on  the  gauze.  By  a 
series  of  such  experiments  Redi  destroyed  the  belief  in  the 
spontaneous  generation  of  maggots  in  meat,  and  with  it 
many  related  beliefs." 

In  1683  Anthony  van  Leeuwenhoek,  justly  called  the 
"Father  of  microscopy,"  demonstrated  the  continuity  of 
arteries  and  veins  through  intervening  capillaries,  thus 
affording  ocular  proof  of  Harvey's  discovery  of  the  circula- 
tion of  the  blood;  discovered  bacteria,  seeing  them  first  in 
saliva,  discovered  the  rotifers,  and  first  saw  the  little  glob- 
ules in  yeast  which  Latour  and  Schwann  subsequently 
proved  to  be  plants. 

Leeuwenhoek  involuntarily  reopened  the  old  contro- 
versy about  spontaneous  generation  by  bringing  forward  a 
new  world,  peopled  by  creatures  of  such  extreme  minuteness 
as  to  suggest  not  only  a  close  relationship  to  the  ultimate 
molecules  of  matter,  but  an  easy  transition  from  them. 

In  succeeding  years  the  development  of  the  compound 
microscope  showed  that  putrescent  infusions,  both  animal 
and  vegetable,  teemed  with  minute  living  organisms. 
*  See  Tyndall:  "  Floating  Matter  in  the  Air." 


The  History  of  the  Subject  19 

Abbe"  Lazzaro  Spallanzani  (1777)  filled  flasks  with  organic 
infusions,  sealed  their  necks,  and,  after  subjecting  their  con- 
tents to  the  temperature  of  boiling  water,  placed  them  under 
conditions  favorable  for  the  development  of  life,  without, 
however,  being  able  to  produce  it.  Spallanzani's  critics, 
however,  objected  to  his  experiment  on  the  ground  that  air 
is  essential  to  life,  and  that  in  his  flasks  the  air  was  excluded 
by  the  hermetically  sealed  necks. 

Schulze  (1836)  set  this  objection  aside  by  filling  a  flask 
only  half  full  of  distilled  water,  to  which  animal  and  vege- 
table matters  were  added,  boiling  the  contents  to  destroy 
the  vitality  of  any  organisms  which  might  already  exist  in 
them,  then  sucking  daily  into  the  flask  a  certain  amount  of 
air  which  was  passed  through  a  series  of  bulbs  containing 
concentrated  sulphuric  acid,  in  which  it  was  supposed  that 
whatever  germs  of  life  the  air  might  contain  would  be  de- 
stroyed. This  flask  was  kept  from  May  to  August ;  air  was 
passed  through  it  daily,  yet  without  the  development  of  any 
infusorial  life. 

It  must  have  been  a  remarkably  germ-free  atmosphere  in 
which  Schulze  worked,  for,  as  was  shown  by  those  who  re- 
peated his  experiment,  under  the  conditions  that  he  regarded 
as  certainly  excluding  all  life,  germs  can  readily  enter  with 
the  air. 

In  1838  Ehrenberg  devised  a  system  of  classifying  the 
minute  forms  of  life,  a  part  of  which,  at  least,  is  still  recog- 
nized at  the  present  time. 

The  term  "infusorial  life"  having  been  used,  it  is  well  to 
remark  that  during  all  the  early  part  of  their  recognized 
existence  the  bacteria  were  regarded  as  animal  organisms 
and  classed  among  the  infusoria. 

Tyndall,  stimulated  by  the  work  of  Pasteur,  conclusive- 
ly proved  that  the  micro-organismal  germs  were  in  the 
dust  suspended  in  the  atmosphere,  and  not  ubiquitous  in 
distribution.  His  experiments  were  very  ingenious  and 
are  of  much  interest.  First  preparing  light  wooden  cham- 
bers, with  a  large  glass  window  in  the  front  and  a  smaller 
window  in  each  side,  he  arranged  a  series  of  test-tubes  in 
the  bottom,  half  in  and  half  out  of  the  chamber,  and  a 
pipet,  working  through  a  rubber  diaphragm,  in  the  top,  so 
that  when  desired  the  tubes,  one  by  one,  could  be  filled 
through  it.  Such  chambers  were  allowed  to  stand  until  all 
the  contained  dust  had  settled,  and  then  submitted  to  an 


20  Introduction 

optical  test  to  determine  the  purity  of  the  contained  atmos- 
phere by  passing  a  powerful  ray  of  light  through  the  side 
windows.  When  viewed  through  the  front,  this  ray  was  vis- 
ible only  so  long  as  there  were  particles  suspended  in  the  at- 
mosphere to  reflect  it.  When  the  dust  had  completely 
settled  and  the  light  ray  had  become  invisible  because  of 
the  purity  of  the  contained  atmosphere,  the  tubes  were 
cautiously  filled  with  urine,  beef-broth,  and  a  variety  of 
animal  and  vegetable  broths,  great  care  being  taken  that 
in  the  manipulation  the  pipet  should  not  disturb  the  dust. 
Their  contents  were  then  boiled  by  submergence  in  a  pan  of 
hot  brine  placed  beneath  the  chamber,  in  contact  with  the 
projecting  ends  of  the  tubes,  and  subsequently  allowed  to 
remain  undisturbed  for  days,  weeks,  or  months.  In  nearly 
every  case  life  failed  to  develop  in  the  infusions  after  the 
purity  of  the  atmosphere  was  established. 

II.  CHEMIC  CONTRIBUTIONS?  FERMENTATION  AND  PUTREFACTION. 

As  in  the  world  of  biology  the  generation  of  life  was  an 
all-absorbing  problem,  so  in  the  world  of  chemistry  the 
phenomena  of  fermentation  and  putrefaction  were  inex- 
plicable so  long  as  the  nature  of  the  ferments  was  not 
understood. 

In  the  year  1837  Latour  and  Schwann  succeeded  in 
demonstrating  that  the  minute  oval  bodies  which  had  been 
observed  in  yeast  since  the  time  of  Leeuwenhoek  were 
living  organisms — vegetable  forms — capable  of  growth. 

So  long  as  yeast  was  looked  upon  as  an  inert  substance 
it  was  impossible  to  understand  how  it  could  impart  fer- 
mentation to  other  substances;  but  when  it  was  shown 
by  Latour  that  the  essential  element  of  yeast  was  a  growing 
plant,  the  phenomenon  became  a  perfectly  natural  conse- 
quence of  life.  Not  only  the  alcoholic,  but  also  the  acetic, 
lactic,  and  butyric  fermentations  have  been  shown  to  re- 
sult from  the  energy  of  low  forms  of  vegetable  life,  chiefly 
bacterial  in  nature.  Prejudice,  however,  prevented  many 
chemists  from  accepting  this  view  of  the  subject,  and 
Liebig  strenuously  adhered  to  his  theory  that  fermenta- 
tion was  the  result  of  the  internal  molecular  movements 
which  a  body  in  the  course  of  decomposition  communicates 
to  other  matter  whose  elements  are  connected  by  a  very 
feeble  affinity. 

Pasteur  was  the  first  to  prove  that  fermentation  is  an 


The  History  of  the  Subject  21 

ordinary  chemic  transformation  of  certain  substances,  tak- 
ing place  as  the  result  of  the  action  of  living  cells,  and 
that  the  capacity  to  produce  it  resides  in  all  animal  and 
vegetable  cells,  though  in  varying  degree. 

In  1862  he  published  a  paper  "On  the  Organized  Cor- 
puscles Existing  in  the  Atmosphere,"  in  which  he  showed 
that  many  of  the  floating  particles  collected  from  the  atmos- 
phere of  his  laboratory  were  organized  bodies.  If  these 
were  planted  in  sterile  infusions,  abundant  crops  of  micro- 
organisms were  obtained.  By  the  use  of  more  refined 
methods  he  repeated  the  experiments  of  others,  and  showed 
clearly  that  ' '  the  cause  which  communicated  life  to  his  in- 
fusions came  from  the  air,  but  was  not  evenly  distributed 
through  it." 

Three  years  later  he  showed  that  the  organized  cor- 
puscles which  he  had  found  in  the  air  were  the  spores  or 
seeds  of  minute  plants,  and  that  many  of  them  possessed 
the  property  of  withstanding  the  temperature  of  boiling 
water — a  property  which  explained  the  peculiar  results 
of  many  previous  experimenters,  who  failed  to  prevent 
the  development  of  life  in  boiled  liquids  inclosed  in  her- 
metically sealed  flasks. 

Chevreul  and  Pasteur,  by  having  proved  that  animal 
solids  do  not  putrefy  or  decompose  if  kept  free  from  the 
access  of  germs,  suggested  to  surgeons  that  putrefaction  in 
wounds  is  due  rather  to  the  entrance  of  something  from 
without  than  to  changes  within.  The  deadly  nature  of  the 
discharges  from  putrescent  wounds  had  been  shown  in  a 
rough  manner  by  Gaspard  as  early  as  1822  by  injecting 
some  of  the  material  into  the  veins  of  animals. 

III.  MEDICAL  AND  SURGICAL  CONTRIBUTIONS,  THE  STUDY  OF  THE 
INFECTIOUS  DISEASES. 

Probably  the  first  writing  in  which  a  direct  relationship 
between  micro-organisms  and  disease  is  suggested  is  by 
Varro,  who  says:  "It  is  also  to  be  noticed,  if  there  be  any 
marshy  places,  that  certain  minute  animals  breed  [there] 
which  are  invisible  to  the  eye,  and  yet,  getting  into  the 
system  through  mouth  and  nostrils,  cause  serious  disorders 
(diseases  which  are  difficult  to  treat)." 

Surgical  methods  of  treatment  depending  for  their  suc- 
cess upon  exclusion  of  the  air,  and  of  course,  incidentally 
if  unknowingly,  exclusion  of  bacteria,  seem  to  have  been 


22  Introduction 

practised  quite  early.  Theodoric,  of  Bologne,  about  1260 
taught  that  the  action  of  the  air  upon  wounds  induced  a 
pathologic  condition  predisposing  to  suppuration.  He  also 
treated  wounds  with  hot  wine  fomentations.  The  wine 
was  feebly  antiseptic,  kept  the  surface  free  from  bacteria, 
and  the  treatment  was,  in  consequence,  a  modification  of 
what  in  later  centuries  formed  antiseptic  surgery. 

Henri  de  Monde ville  in  1306  went  even  further  than 
Theodoric,  whom  he  followed,  and  taught  the  necessity 
of  bringing  the  edges  of  a  wound  together,  covered  it  with 
an  exclusive  plaster  compounded  of  turpentine,  resin,  and 
wax,  and  then  applied  the  hot  wine  fomentation. 

In  1546  Geronimo  Fracastorius  published  at  Venice  a 
work  "  De  contagione  et  contagiosis  morbis  et  curatione,"  in 
which  he  divided  infectious  diseases  into — 

1.  Those  infecting  by  immediate  contact  (true  contagions). 

2.  Those  infecting  through  intermediate  agents,  such  as 
fomites. 

3.  Those  infecting  at  a  distance  or  through  the  air;  he 
mentions  as  belonging  to  this  class  phthisis,  the  pestilential 
fevers,  and  a  certain  kind  of  ophthalmia  (conjunctivitis). 

"  In  his  account  of  the  true  nature  of  disease  germs,  or 
seminaria  contagionum,  ...  he  describes  them  as  particles 
too  small  to  be  apprehended  by  our  senses,  but  as  capable  in 
appropriate  media  of  reproduction,  and  in  this  way  of  in- 
fecting surrounding  tissues. 

"  These  pathogenic  units  Fracastorius  supposed  to  be  of 
the  nature  of  colloidal  systems,  for  if  they  were  not  viscous 
or  glutinous  by  nature  they  could  not  be  transmitted  by  fo- 
mites. Germs  transmitting  disease  at  a  distance  must  be  able 
to  live  in  the  air  a  certain  length  of  time,  and  this  condition 
lie  holds  is  possible  only  when  the  germs  are  gelatinous  or 
colloidal  systems,  for  only  hard,  inert,  discrete  particles 
could  endure  longer. 

"  Fracastorius  conceived  that  the  germs  became  pathogenic 
through  the  action  of  animal  heat,  and  in  order  to  produce 
disease  it  is  not  necessary  that  they  should  undergo  dissolu- 
tion, but  only  metabolic  change."* 

In  1671  Kircher  wrote  a  book  in  which  he  expressed  the 

opinion  that  puerperal  fever,  purpura,  measles,  and  various 

other  fevers  were  the  result  of  a  putrefaction  caused  by 

worms  or  animalcules.     His  opinions  were  thought  by  his 

*"Brit.  Med.  Jour.,"  May  7,  1910,  p.  1122. 


The  History  of  the  Subject  23 

contemporaries  to  be  founded  upon  too  little  evidence,  and 
were  not  received. 

Plencig,  of  Vienna,  became  convinced  that  there  was  an 
undoubted  connection  between  the  microscopic  animal- 
cules exhibited  by  the  microscope  and  the  origin  of  dis- 
ease, and  advanced  this  opinion  as  early  as  1762.  Unfor- 
tunately, his  opinions  seem  not  to  have  been  accepted  by 
others,  and  were  soon  forgotten. 

In  1704  John  Colbach  described  "a  new  and  secret 
method  of  treating  wounds  by  which  healing  took  place 
quickly,  without  inflammation  or  suppuration." 

Boehm  succeeded  in  1838  in  demonstrating  the  occur- 
rence of  yeast  plants  in  the  stools  of  cholera,  and  con- 
jectured that  the  process  of  fermentation  was  concerned 
in  the  causation  of  that  disease. 

In  1840  Henle  considered  all  the  evidence  that  had 
been  collected,  and  concluded  that  the  cause  of  the  infec- 
tious diseases  was  to  be  sought  for  in  minute  living  organ- 
isms or  fungi.  He  may  be  looked  upon  as  the  real  pro- 
pounder  of  the  GERM  THEORY  OF  DISEASE,  for  he  not  only 
collected  facts  and  expressed  opinions,  but  also  investi- 
gated the  subject  ably.  The  requirements  which  he  formu- 
lated in  order  that  the  theory  might  be  proved  were  so 
severe  that  he  was  never  able  to  attain  to  them  with  the 
crude  methods  at  his  disposal.  They  were  so  ably  elabo- 
rated, however,  that  in  after  years  they  were  again  postulated 
by  Koch,  and  it  is  only  by  strict  conformity  with  them 
that  the  definite  relationship  between  bacteria  and  disease 
has  been  determined. 

Briefly  summarized,  these  requirements  are  as  follows: 

1.  A  specific  micro-organism  must  be  constantly  asso- 
ciated with  the  disease. 

2.  It  must  be  isolated  and  studied  apart  from  the  disease. 

3.  When  introduced  into  healthy  animals  it  must  pro- 
duce the  disease,  and  in  the  animal  in  which  the  disease 
has  been  experimentally '  produced  the  organism  must  be 
found  under  the  original  conditions. 

In  1843  Dr.  Oliver  Wendell  Holmes  wrote  a  paper  upon 
the  ''  Contagiousness  of  Puerperal  Fever." 

In  1847  Semmelweiss,  of  Vienna,  struck  by  the  similarity 
between  fatal  wound  infection  with  pyemia  and  puerperal 
fever,  cast  aside  the  popular  theory  that  the  latter  affection 
was  caused  by  the  absorption  into  the  blood  of  milk  from 


24  Introduction 

the  breasts,  and  announced  his  belief  that  the  disease 
depended  upon  poisons  carried  by  the  ringers  of  physicians 
and  students  from  the  dissecting  room  to  the  woman  in 
child-bed,  and  recommended  washing  the  hands  of  the 
accoucheur  with  chlorin  or  chlorid  of  lime,  in  addition  to 
the  use  of  soap  and  water.  He  was  laughed  to  scorn  for 
his  pains. 

In  1849  J.  K.  Mitchell,  in  a  brief  work  upon  the  "Crypto- 
gamous  Origin  of  Malarious  and  Epidemic  Fevers,"  fore- 
shadowed the  germ  theory  of  disease  by  collecting  a  large 
amount  of  evidence  to  show  that  malarial  fevers  were  due 
to  infection  by  fungi. 

Pollender  (1849)  and  Davaine  (1850)  succeeded  in 
demonstrating  the  presence  of  the  anthrax  bacillus  in 
the  blood  of  animals  suffering  from  and  dead  of  that  dis- 
ease. Several  years  later  (1863)  Davaine,  having  made 
numerous  inoculation  experiments,  demonstrated  that  this 
bacillus  was  the  materies  morbi  of  the  disease.  The  bacillus 
of  anthrax  was  probably  the  first  bacterium  shown  to  be 
specific  for  a  disease.  Being  a  very  large  bacillus  and  a 
strongly  vegetative  organism,  its  growth  was  easily  observed, 
while  the  disease  was  one  readily  communicated  to  animals. 

Klebs,  who  was  one  of  the  pioneers  of  the  germ  theory, 
published,  in  1872,  a  work  upon  septicemia  and  pyemia, 
in  which  he  expressed  himself  convinced  that  the  causes 
of  these  diseases  must  come  from  without  the  body.  Bill- 
roth,  however,  strongly  opposed  such  an  idea,  asserting 
that  fungi  had  no  especial  importance  either  in  the  processes 
of  disease  or  in  those  of  decomposition,  but  that,  existing 
everywhere  in  the  air,  they  rapidly  developed  in  the  body 
as  soon  as  through  putrefaction  a  "  Faulnisszymoid  "  (putre- 
factive ferment),  or  through  inflammation  a  "Phlogisti- 
schezymoid"  (inflammatory  ferment),  supplying  the  neces- 
sary feeding-grounds,  was  produced. 

In  1873  Obermeier  observed  that  actively  motile,  flexible 
spiral  organisms  were  present  in  large  numbers  in  the 
blood  of  patients  in  the  febrile  stages  of  relapsing  fever.  . 

In  1875  the  number  of  scientific  men  who  had  entirely 
abandoned  the  doctrine  of  spontaneous  generation  and 
embraced  the  germ  theory  of  disease  was  small,  and  most 
of  those  who  accepted  it  were  experimenters.  A  great 
majority  of  medical  men  either  believed,  like  Billroth,  that 
the  presence  of  fungi  where  decomposition  was  in  progress 


The  History  of  the  Subject  25 

was  an  accidental  result  of  their  universal  distribution,  or, 
being  still  more  conservative,  adhered  to  the  old  notion 
that  the  bacteria,  whose  presence  in  putrescent  wounds  as 
well  as  in  artificially  prepared  media  was  unquestionable, 
were  spontaneously  generated  there. 

Before  many  of  the  important  bacteria  had  been  dis- 
covered, and  while  ideas  upon  the  relation  of  micro-organ- 
isms to  disease  were  most  crude,  some  practical  measures 
were  suggested  that  produced  greater  agitation  and  incited 
more  observation  and  experimentation  than  anything  sug- 
gested in  surgery  since  the  introduction  of  anesthetics — 
namely,  antisepsis. 

"It  is  to  one  of  old  Scotia's  sons,  Sir  Joseph  Lister, 
that  the  everlasting  gratitude  of  the  world  is  due  for  the 
knowledge  we  possess  in  regard  to  the  relation  existing 
between  micro-organisms  and  inflammation  and  suppura- 
tion, and  the  power  to  render  wounds  aseptic  through 
the  action  of  germicidal  substances."  * 

Lister,  convinced  that  inflammation  and  suppuration 
were  due  to  the  entrance  of  germs  from  the  air,  instru- 
ments, fingers,  etc.,  into  wounds,  suggested  the  employ- 
ment of  carbolic  acid  for  the  purpose  of  keeping  sterile 
the  hands  of  the  operator,  the  skin  of  the  patient,  the 
surface  of  the  wound,  and  the  instruments  used.  He 
finally  concluded  every  operation  by  a  protective  dressing 
to  exclude  the  entrance  of  germs  at  a  subsequent  period. 

Listerism,  or  "antisepsis,"  originated  in  1875,  and  when 
Koch  published  his  famous  work  on  the  "Wundinfections- 
krankheiten"  (traumatic  infectious  diseases),  in  1878,  it 
spread  slowly  at  first,  but  surely  in  the  end,  to  all  depart- 
ments of  surgery  and  obstetrics. 

From  time  to  time,  as  the  need  for  them  was  realized,  the 
genius  of  investigators  provided  new  devices  which  materially 
aided  in  their  work,  and  have  made  possible  many  discoveries 
that  must  otherwise  have  failed.  Among  them  may  be  men- 
tioned the  improvement  of  the  compound  microscope,  the  use 
of  sterilized  culture  fluids  by  Pasteur,  the  introduction  of 
solid  culture  media  and  the  isolation  methods  by  Koch, 
the  use  of  the  cotton  plug  by  Schroeder  and  van  Dusch, 
and  the  introduction  of  the  anilin  dyes  by  Weigert. 

It  is  interesting  to  note  that  after  the  discovery  of  the 
anthrax  bacillus  by  Pollender  and  Davaine,  in  1849,  there 
*Agnew's  "Surgery,"  vol.  i,  chap.  11. 


26  Introduction 

was  a  period  of  nearly  twenty-five  years  during  which  no 
important  pathogenic  organisms  were  discovered,  but  during 
which  technical  methods  were  being  elaborated,  making 
possible  a  rapid  succession  of  subsequent  important  dis- 
coveries. 

Thus,  in  1873,  Obermeier  discovered  Spirillum  ober- 
meieri  of  relapsing  fever. 

In  1879  Hansen  announced  the  discovery  of  bacilli  in 
the  cells  of  leprous  nodules,  and  Neisser  discovered  the 
gonococcus. 

In  1880  the  bacillus  of  typhoid  fever  was  observed  by 
Hberth  and  independently  by  Koch,  Pasteur  published  his 
work  upon  "Chicken-cholera,"  and  Sternberg  described  the 
pneumococcus,  calling  it  Micrococcus  pasteuri. 

In  1882  Koch  made  himself  immortal  by  his  discovery 
of  and  work  upon  the  tubercle  bacillus,  and  in  the  same  year 
Pasteur  published  a  work  upon  "Rouget  du  Pore,"  and 
Loffler  and  Shiitz  discovered  the  bacillus  of  glanders. 

In  1884  Koch  reported  the  discovery  of  the  "comma 
bacillus,"  the  cause  of  cholera,  and  in  the  same  year  Loffler 
isolated  the  diphtheria  bacillus,  and  Nicolaier  the  tetanus 
bacillus. 

In  1892  Canon  and  Pfeiffer  discovered  the  bacillus  of 
influenza. 

In  1894  Yersin  and  Kitasato  independently  isolated  the 
bacillus  causing  the  bubonic  plague,  then  prevalent  at 
Hong-Kong. 

A  new  era  in  bacteriology,  and  probably  the  most  tri- 
umphant achievement  of  scientific  medicine,  was  inau- 
gurated in  1890,  when  Behring  discovered  the  principles  of 
the  "  blood-serum  therapy."  Since  that  time  investigations 
have  been  largely  along  the  lines  of  immunity,  immunization, 
and  the  therapeutic  serums,  the  names  of  Behring,  Kitasato, 
Wernicke,  Roux,  Khrlich,  Metschnikoff,  Bordet,  Wasser- 
mann,  Shiga,  Madsen,  and  Arrhenius  taking  front  rank. 

The  discovery  of  the  Treponema  pallidum,  the  specific 
organism  of  syphilis,  was  made  in  1905  by  Schaudinn  and 
Hoffmann,  long  after  clinical  study  of  the  disease  had  antici- 
pated it  to  such  an  extent  that  when  the  discovery  was  finally 
made  it  was  unnecessary  to  modify  our  ideas  of  the  disease  in 
any  essential. 

In  the  same  year,  1905,  Castellani  discovered  the  Trepo- 
nema pertenue,  the  cause  of  frambesia  or  yaws. 


The  History  of  the  Subject  27 

In  1911  Noguchi  succeeded  in  obtaining  pure  cultures  of 
the  treponema. 

During  the  time  that  so  much  investigation  of  the  prob- 
lems of  infection  was  in  progress  the  discoveries  were  by  no 
means  restricted  to  the- bacteria  and  their  products,  as  the 
reader  might  infer  from  the  perusal  of  a  chapter  whose  pur- 
pose is  to  explain  the  development  of  the  department  of 
science  now  known  as  Bacteriology.  Other  organisms  of 
different — i.  e.,  animal — nature  were  also  found  in  large 
numbers. 

In  1875  Losch  discovered  the  Amoeba  coli;  in  1878  Rivolta 
described  the  Coccidium  cuniculi  of  the  rabbit;  in  1879 
Lewis  first  saw  Trypanosoma  lewisi  in  the  blood  of  the  rat; 
in  1 88 1  Laveran  discovered  Plasmodium  malariae  in  the  blood 
of  cases  of  human  paludism;  in  1885  Blanchard  described 
the  sarcocystis  in  muscle-fibers;  in  1893  Councilman  and 
Lafleur  studied  Amoeba  dysenteriae  in  the  stools  and  tissues 
of  human  dysentery;  in  1903  Leishman  and  Donovan  found 
the  little  body  Leishmania  donovani  in  the  splenic  juice  of 
cases  of  kala-azar,  and  in  1903  Dutton  and  Forde,  working 
independently,  observed  trypanosomes — the  Trypanosoma 
gambienseof  African  lethargy — in  the  blood  of  human  beings. 

Each  of  these  was  followed  by  new  discoveries  and  addi- 
tions in  its  own  sphere,  and  the  systematic  consideration  of 
these  protozoan  organisms  can  only  be  undertaken  in  a  text- 
book devoted  to  animal  parasites. 

Many  of  these  organisms,  however,  can  be  cultivated  and 
studied  by  the  methods  of  bacteriology,  and,  indeed,  it  is  the 
progress  of  bacteriology  that  has  made  our  ever-increasing 
knowledge  of  them  possible. 

That  the  specific  micro-organisms  of  many  of  the  infectious 
diseases  remained  undiscovered  was  a  source  of  perplexity 
so  long  as  it  was  supposed  that  all  living  things  must  be  visible 
to  the  eye  aided  by  the  microscope.  To-day,  thanks  to  the 
invention  of  the  ultramicroscope,  that  shows  the  existence 
of  things  too  small  to  be  defined,  and  still  more  to  the  adap- 
tation of  the  method  of  filtration  to  the  study  of  the  diseases  in 
question,  we  realize  that  the  "  viruses  "  of  disease  may  be  vis- 
ible or  invisible  and  that  they  have  no  limitations  of  size. 
Just  as  bacteria  readily  find  their  way  through  paper  filters, 
so  the  invisible  and  hence  undescribed  viruses — i.  e.,  micro- 
organisms— of  rabies,  poliomyelitis,  yellow  fever,  pleuro- 
pneumonia  of  cattle,  foot-and-mouth  disease,  rinderpest, 


28  Introduction 

hog-cholera,  African  horse-fever,  infectious  anemia  or  swamp 
sickness  of  horses,  fowl  plague,  small-pox,  cow-pox,  sheep- 
pox,  horse-pox,  swine-pox,  and  goat-pox  are  at  some  or  all 
stages  able  to  pass  through  the  Berkefeld  or  diatomaceous 
earth  filters,  and  some  of  them  through  the  much  less  porous 
unglazed  porcelain  or  Chamberland  filters.  Thus  there  is 
opened  a  new  world  that  is  ultramicroscopic,  but  still  teems 
with  invisible  living  organisms. 

Such  organisms,  interesting  as  they  must  be  to  the  biolo- 
gist and  pathologist,  cannot  yet  be  known  or  described,  and, 
therefore,  seem  to  be,  for  the  most  part,  beyond  the  scope  of 
the  present  writing. 


CHAPTER   I. 

STRUCTURE  AND  CLASSIFICATION  OF  THE 
MICRO-ORGANISMS. 

BACTERIA. 

WHEN  Leeuwenhoek  with  his  improved  microscope  dis- 
covered the  new  world  of  micro-organisms,  he  supposed  them, 
on  account  of  the  active  movements  they  manifested,  to  be 
small  animals,  and  described  them  as  animalculae.  The 
early  systematic  writers,  Ehrenberg  and  Dujardin,  fell  into 
the  same  error,  and  it  was  many  years  before  biologists  had 
arrived  at  even  approximate  accuracy  in  arranging  them. 
Indeed,  for  a  long  time  a  great  number  baffled  systematic 
writers,  and  no  less  an  authority  than  Haeckel,  in  1878,  sug- 
gested that  they  form  a  group  by  themselves  to  be  known  as 
Protista.  Such  a  grouping,  however,  was  unsatisfactory 
alike  to  botanists  and  zoologists,  and,  therefore,  was  used  by 
few. 

It  was  evident  that  structure  could  not  be  looked  upon  as 
a  satisfactory  differential  character,  for  between  the  protozoa, 
or  most  simple  animals,  and  the  protophyta,  or  most  simple 
plants,  the  structural  differences  were  too  minute  to  prevent 
overlapping.  Motion  and  locomotion  had  to  be  abandoned, 
since  it  was  common  to  both  groups.  Reproduction  was 
likewise  an  unreliable  means  when  taken  by  itself,  for  much 
the  same  means  of  multiplication  were  found  to  obtain  in 
both  groups.  One  great  physiologic  and  metabolic  differ- 
ence was,  however,  noted:  plants  possess  the  power  of 
nourishing  themselves  upon  purely  inorganic  compounds, 
while  animals  are  unable  to  do  so  and  cannot  live  except 
upon  complex  molecular  combinations  synthesized  by  the 
plants.  In  this  metabolic  difference  we  find  the  present 
criterion  for  the  separation  of  the  living  organisms  into  the 
two  main  groups!  But  this  does  not  dispose  of  all  of  the 
difficulties,  for  there  are  certain  small  groups  to  which  it 

29 


30    Structure  and  Classification  of  Micro-organisms 

does  not  apply.  Thus,  for  example,  the  fungi  which,  when 
judged  by  other  criteria,  are  undoubted  plants,  lack  the  power 
of  inorganic  synthesis,  and  so  resemble  animals. 

Fortunately,  the  question  is  a  purely  academic  one.  Though 
seemingly  at  first  sight  a  most  fundamental  one,  it  is,  in 
reality,  of  trifling  importance,  for  after  a  limited  experience 
the  student  unhesitatingly  assigns  most  of  the  known  or- 
ganisms to  one  or  the  other  groups,  and  that  occasional  mis- 
takes may  be  made,  and  organisms,  like  the  spirochaeta, 
appear  sometimes  in  the  group  of  plants  among  the  bacteria, 
and  in  other  writings  among  the  protozoa,  is  a  matter  of  small 
consequence  so  long  as  the  knowledge  of  the  organisms  them- 
selves is  in  no  particular  diminished  by  the  method  of  classi- 
fication. 

In  discussing  the  matter  Delage  says,  ' '  The  question  is  not 
so  important  as  it  appears.  From  one  point  of  view  and  on 
purely  theoretic  grounds  it  does  not  exist,  while  from  an- 
other standpoint  it  is  insoluble.  If  one  be  asked  to  divide 
living  things  into  two  distinct  groups,  of  which  one  contains 
only  animals  and  the  other  only  plants,  the  question  is  mean- 
ingless, for  plants  and  animals  are  concepts  which  have  no 
objective  reality,  and  in  nature  they  are  only  individuals. 
If  in  considering  those  forms  which  we  regard  as  true  ani- 
mals and  plants  we  look  for  their  phylogenetic  history  and 
decide  to  place  all  of  their  allies  in  one  or  the  other  group, 
we  are  sure  to  reach  no  result;  such  attempts  have  always 
been  fruitless." 

"  Huxley  pointed  out  as  early  as  1876  the  extremely  close 
relationship  between  the  lowest  algae  and  some  of  the  flagel- 
lates, and  it  is  the  general  opinion  that  no  one  feature  sep- 
arates the  lowest  plants  from  the  lowest  animals,  and  the 
difficulty — in  many  cases  the  impossibility — of  distinguishing 
between  them  is  clearly  recognized. 

'  The  point  of  view  which  demands  a  strict  separation  of 
animals  and  plants  has,  however,  little  utility  save,  perhaps, 
to  determine  the  limits  of  a  text-book  or  a  monograph."* 

The  relative  position  of  the  pathogenic  vegetable  micro- 
organisms to  the  other  vegetable  organisms  can  be  deter- 
mined by  reference  to  the  following  table.  The  wide  sepa- 
ration of  the  bacteria  in  Group  II.  and  all  of  the  others, 
which  appear  in  Group  X.,  should  be  noted. 

*  Calkin's,  "The  Protozoa,"  p.  23. 


Bacteria  31 


TABLE  I. 

THE  PLANT  KINGDOM. 


'"d  These  primary  divisions,  like  the  cor- 

3  If  responding  primary  division  of  animals 

.£5  ^  into   vertebrata  and   invertebrata,   are 

g-2  now  falling  into  disuse 


s. 
«fr 

IF 


!] 


X 


W  W     £     W3QON03  Jf 

iv         ii  iirfiii Qg,f 


~        T          o 

! 


06  E-d-g-FE: 

"Ilfp 


The  various  genera  to  which  the  pathogenic  fungi  belong 
are  by  no  means  closely  related  to  one  another,  as  can  at 
once  be  seen  by  the  following  amplification  of  group  X. 
Eumycetes : 


32     Structure  and  Classification  of  Micro-organisms 

TABLE  II. 

X.  Eumycetes  (ev  good,  MW"?TOS  fungus).    The  true  fungi:  plants  without  chlorophyl. 
Class  i.  Phycomycetes  (<t>vKos  seaweed),  alga-like  fungi. 
Order  i.  Zygomycetes. 

Sub-order — Mucorineae. 
Family — Mucoraceae. 
Genus — Mucor. 
Order  2.  Oomycetes. 

Class  2.  Hemiascomycetes. 
Order  i.  Hemiascales. 

Family — Saccharomycetaceae. 
Genus — Saccharomyces . 
"    —  Blastomyces  (?). 


Class  3.  Euascomycetes. 

Order  i.  Euascales  (contains  45  families). 
Family — Aspergilloceae. 
Genus— Aspergillus. 
"    — Penicillium. 


Fungi  imperfecti. 
This  is  a  large  supplementary 
group,  of  imperfectly  known 
fungi    not    included    in    the 
tabulation. 
In  it  we  find  Oidium. 


Class  4.  Laboulbeniomycetes. 
Order  i.  Laboulbeniales. 

Class  5.  Basidiomycetes. 
Sub-class — Hemibasidii. 

Order  i.  Hemibasidiales. 

Family — Ustilaginaceae  (smuts). 
Sub-class — Eubasidii. 

Order  i.  Protobasidiomycetes. 

Family— Uredineineae  (rusts). 
Order  2.  Autobasidiomycetes  (mushrooms,  toad-stools,  etc.). 

No  entirely  satisfactory  grouping  of  the  bacteria  themselves 
has  yet  been  achieved,  the  best  characters  to  be  used  as 
the  basis  of  classification  being  undecided.  The  best  system 
for  their  provisional  arrangement  is  probably  that  of  Migula,* 
or  the  modification  of  it  suggested  by  F.  D.  Chester,  f  in 
which  the  morphology,  sporulation,  and  appendages  of  the 
bacteria  all  enter  as  important  features. 

CLASSIFICATION  OF  THE  BACTERIA. 
I.  ORDER:  EUBACTERIA  (True  Bacteria). 

A.  SUB-ORDER:  Haplobacteria  (Lower  Bacteria). 
I.  Family   COCCACE;E.     Cells  globular,   becoming  slightly  elongate 
before  division.     Division  in  one,  two,  or  three  directions  of 
space.     Formation  of  endospores  very  rare. 
(A)  Without  flagella. 

1.  Streptococcus.     Division  in  one  direction  of  space,  producing 

chains  like  strings  of  beads. 

2.  Micrococcus.     Division  in  two  directions  of  space,  so  that 

tetrads  are  often  formed. 

3.  Sarcina.     Division  in  three  directions  of  space,  leading  to  the 

formation  of  bale-like  packages. 

*  "System  der  Bakterien,"  Jena,  1897-1900  (vols.  I  and  II  appearing 
at  different  times). 

f  "Preliminary  Arrangement  of  the  Species  of  the  Genus  Bacterium," 
"Ninth  Annual  Report  of  the  Delaware  College  Agricultural  Experi- 
ment Station,"  1897,  Newark,  Delaware,  U.  S.  A. 


Bacteria  33 

(B)  Withflagella. 

1.  Planococcus.     Division  in  two  directions  of  space,  like  micro- 

coccus. 

2.  Planosarcina.     Division  in  three  directions,  like  sarcina. 

II.  Family  BACTERIACE^E.  Cells  more  or  less  elongate,  cylindric,  and 
straight.  They  never  form  spiral  windings.  Division  in  one 
direction  of  space  only,  transverse  to  the  long  axis  of  the  cell. 

(A)  Without  flagella. 

1.  Bacterium.     Occasional  endospores. 

(B)  Withflagella. 

2.  Bacillus.     Flagella    arising   from   any    part   of   the    surface. 

Endospore-formation  common. 

3.  Pseudomonas.     Flagella  attached  only  at  the  ends  of  the  cell. 

Endospores  very  rare. 

III.  Family  SPIRILLACE^E.  Cells  twisted  spirally  like  a  corkscrew,  or 
representing  sections  of  the  spiral.  Division  only  transverse 
to  the  long  diameter. 

1.  Spirosoma.     Rigid;  without  flagella. 

2.  Microspira.     Rigid;   having   one,   two,   or  three   undulating 

flagella  at  the  ends. 

3.  Spirillum.     Rigid;  having  from  five  to  twenty  curved  or  un- 

dulating flagella  at  the  ends. 

4.  SpirochfBta*     Serpentine  and  flexible.     Flagella  not  observed ; 

probably  swim  by  means  of  an  undulating  membrane. 

B.  SUB-ORDER:  Trichobacteria  (Higher  Bacteria). 

IV.  Family  MYCOBACTERIACE^S.  Cells  forming  long  or  short  cylindric 
filaments,  often  clavate-cuneate  or  irregular  in  form,  and  at 
times  showing  true  or  false  branchings.  No  endospores,  but 
formation  of  gonidia-like  bodies  due  to  segmentation  of  the  cells. 
No  flagella.  Division  at  right  angles  to  the  axis  of  rod  in  fila- 
ment. Filaments  not  surrounded  by  a  sheath  as  in  Chlamydo- 
bacteriaceae. 

1.  Mycobacterivm.     Cells  in  their  ordinary  form,  short  cylindric 

rods  often  bent  and  irregularly  cuneate.  At  times  Y- 
shaped  forms  or  longer  filaments  with  true  branchings 
may  produce  short  coccoid  elements,  perhaps  gonidia. 
(This  genus  includes  the  Corynebacterium  of  Lehmann- 
Neumann.)  No  flagella. 

2.  Actinomyces.     Cells  in  their  ordinary  form  as  long  branched 

filaments;  growth  coherent,  dry  or  crumpled.  Produce 
gonidia-like  bodies.  Cultures  generally  have  a  moldy 
appearance,  due  to  the  development  of  aerial  hyphae.  No 
flagella. 

V.  Family  CHLAMYDOBACTERIACE^.     Forms  that  vary  in  different 
stages  of  their  development,  but  all  characterized  by  a  sur- 
rounding sheath  about  both  branched  and  unbranched  threads. 
Division  transverse  to  the  length  of  the  filaments, 
i.   Cladothrix.     Characterized   by   pseudo-dichotomous   branch- 
ings.    Division    only    transverse.     Multiplication    by    the 
separation  of  whole  branches.     Transplantation  by  means 
of  polar  flagellated  swarm-spores. 

*  The  spirochaeta  and  some  closely  related  forms  are  now  thought  to 
be  more  properly  classified  among  the  protozoa  than  among  the  bac- 
teria. They  will,  therefore,  appear  again  in  the  tabulation  of  the 
protozoan  organisms. 

3 


34    Structure  and  Classification  of  Micro-organisms 

2.  Crenothrix.     Cells  united  to  form  unbranched  threads  which 

in  the  beginning  divide  transversely.  Later  the  cells 
divide  in  all  three  directions  of  space.  The  products  of 
final  division  become  spheric,  and  serve  as  reproductive 
elements. 

3.  Phragmidiothrix.     Cells    at    first    united    into    unbranched 

threads.  Divide  in  three  directions  of  space.  Late  in  the 
development,  by  the  growth  of  certain  of  the  cells  through 
the  delicate,  closely  approximated  sheath,  branched  forms 
are  produced. 

4.  Thiothrix.     Unbranched  cells  inclosed  in  a  delicate  sheath. 

Non-motile.  Division  in  one  direction  of  space.  Cells 
contain  sulphur  grains. 

II.  ORDER:  THIOBACTERIA   (Sulphur  Bacteria). 

I.  Family  BEGGIATOACE.£.  Cells  united  to  form  threads  which  are 
not  surrounded  by  an  inclosing  sheath.  The  septa  are  scarcely 
visible.  Divide  in  one  direction  of  space  only.  Motility  ac- 
complished through  the  presence  of  an  undulating  membrane. 
Cells  contain  sulphur  grains. 

There  are  two  families,  numerous  subfamilies,  and  thirteen  genera  in 
this  order.  They  are  all  micro-organisms  of  the  water  and  soil,  and 
have  no  interest  for  the  medical  student. 

Structure. — Nucleus. — When  subjected  to  the  action  of 
nuclear  stains,  large  vague  nuclear  formations  are  usually 
observed  in  the  bacterial  cells. 

Cytoplasm. — The  cytoplasm,  of  which  very  little  exists 
between  the  large  nucleus  and  cell- wall,  is  sometimes  granu- 
lar, as  in  Bacillus  megatherium,  and  sometimes  contains  fine 
granules  of  chlorophyl,  sulphur,  fat,  or  pigment. 

Capsule. — Each  cell  is  surrounded  by  a  distinct  cell- wall, 
which  in  some  species  shows  the  cellulose  reaction  with 
iodin. 

The  cell-walls  of  certain  bacteria  at  times  undergo  a 
peculiar  gelatinous  change  or  permit  the  exudation  of 
gelatinous  material  from  the  cytoplasm,  and  appear  sur- 
rounded by  a  halo  or  capsule.  Such  capsules  are  seen  about 
the  pneumococcus  as  found  in  blood  or  sputum,  Friedlander's 
bacillus,  as  seen  in  sputum,  Bacillus  aero  genes  capsulatus  in 
blood  or  tissue,  and  many  other  organisms.  Friedlander 
pointed  out  that  the  capsule  of  his  pneumonia  bacillus,  as 
found  in  the  lung  tissue  or  in  the  "prune-juice"  sputum, 
was  very  distinct,  though  it  could  not  be  demonstrated  at 
all  when  the  organisms  grew  in  gelatin. 

Polar  Granules. — By  carefully  staining  an  appropriate 
organism  certain  peculiarities  of  structure  can  sometimes 
be  shown.  Thus,  some  bacilli  contain  distinct  "  polar  gran- 
ules" (metachromatic  or  Babes-Ernst  granules) — rounded  or 


Bacteria  35 

oval  bodies — situated  at  the  ends  of  the  cell.  Their  sig- 
nificance is  unknown.  They  have  been  supposed  to  bear 
some  relationship  to  the  biologic  activity  of  the  organ- 
ism, especially  its  pathogenesis,  but  this  is  uncertain,  and 
a  recent  work  by  Gauss*  and  Schumburgf  shows  that 
they  vary  with  the  reaction  of  the  culture-media  upon  which 
the  bacteria  grow  and  have  nothing  to  do  with  their  viru- 
lence. Bacillus  megatherium  is  peculiar  in  having  its 
cytoplasm  filled  with  small  granules  which  stain  deeply. 
The  diphtheria  bacillus  and  the  cholera  spirillum  stain  very 
irregularly  in  fresh  cultures,  as  if  the  tingeable  substance 
were  not  uniformly  distributed  throughout  the  cytoplasm. 
Vacuolated  bacteria  and  bacteria  that  will  not  stain,  or  stain 
very  irregularly,  may  usually  be  regarded  as  degenerated 
organisms  (involution  forms)  which,  because  of  plasmolysis, 
or  solution,  can  no  longer  stain  homogeneously. 

Flagella. — Many  bacteria  possess  delicate  straight  or  wavy 
filaments,  called  flagella,  which  appear  to  be  organs  of  loco- 
motion. 

Messeat  has  suggested  that  the  bacteria  be  classified,  ac- 
cording to  the  arrangement  of  the  flagella,  into : 

I.  Gymnobacteria  (forms  without  flagella). 
II.  Trichobacteria  (forms  with  flagella). 

1.  Monotricha  (with  a  single  flagellum  at  one  end). 

2.  Lophotrocha  (with  a  bundle  of  flagella  at  one  end). 

3.  Amphitricha  (with  a  flagellum  at  each  end). 

4.  Peritricha  (flagella  around  the  body,  springing  from  all 

parts  of  its  surface). 

This  arrangement  is-,  however,  less  satisfactory  than  that 
of  Migula  already  given. 

Motility. — The  greater  number  of  the  bacteria  supplied 
with  flagella  are  actively  motile,  the  locomotory  power  no 
doubt  being  the  lashing  flagella.  The  rod  and  spiral  micro- 
organisms are  most  plentifully  supplied  with  flagella;  only 
a  few  of  the  spheric  forms  have  them. 

The  presence  of  flagella,  however,  does  not  invariably  im- 
ply motility,  as  they  may  also  serve  to  stimulate  the  passage 
of  currents  of  nutrient  fluid  past  the  organism,  and  so  favor 
its  nutrition.  The  flagellate  bacteria  are  more  numerous 
among  the  saprophytic  than  the  pathogenic  forms. 

*  "Centralbl.  f.  Bakt.,"  etc.,  xxxi,  No.  3,  Feb.  5,  1902,  p.  106. 

f  Ibid.,  xxxi,  No.  14,  p.  694,  June  3,  1902. 

J  "Rivista  d'igiene  e  sanata  publica,"  1890,  n. 


36    Structure  and  Classification  of  Micro-organisms 

Bacillus  megatherium  has  a  distinct  but  limited  ameboid 
movement. 

The  dancing  movement  of  some  of  the  spheric  bacteria  seems  to  be 
the  well-known  Brownian  movement,  which  is  a  physical  phenomenon. 
It  is  sometimes  difficult  to  determine  whether  an  organism  viewed  under 
the  microscope  is  really  motile  or  whether  it  is  only  vibrating.  One 
can  usually  determine  by  observing  that  in  the  latter  case  it  does  not 
change  its  relative  position  to  surrounding  objects. 

In  some  cases  the  colonies  of  actively  motile  bacteria, 
such  as  the  proteus  bacilli,  show  definite  migratory  tenden- 
cies upon  5  per  cent,  gelatin.  The  active  movement  of 
the  bacteria  composing  the  colony  causes  its  shape  con- 
stantly to  change,  so  that  it  bears  a  faint  resemblance  to  an 
ameba,  and  moves  about  from  place  to  place  upon  the  sur- 
face of  the  gelatin. 

Size. — Bacteria  are  so  minute  that  a  special  unit  has 
been  adopted  for  their  measurement.  This  is  the  micro- 
millimeter  (//),  or  one-thousandth  part  of  a  millimeter, 
equivalent  to  the  one-twenty-five-thousandth  (-jTriinr)  of  an 
inch. 

The  size  of  bacteria  varies  from  a  fraction  of  a  micro- 
millimeter  to  20  or  even  40  micromillimeters. 

Reproduction. — Fission. — Bacteria  multiply  by  binary 
division  (fission).  A  bacterium  about  to  divide  appears 
larger  than  normal,  and,  if  a  spheric  organism,  more  or  less 
ovoid.  By  appropriate  staining  karyokinetic  changes  may 
be  observed  in  the  nuclei.  When  the  conditions  of  nutri- 
tion are  good,  fission  progresses  with  astonishing  rapidity. 
Buchner  and  others  have  determined  the  length  of  a  gener- 
ation to  be  from  fifteen  to  forty  minutes. 

The  results  of  binary  division,  if  rapidly  repeated,  are 
almost  appalling.  "Cohn  calculated  that  a  single  germ 
could  produce  by  simple  fission  two  of  its  kind  in  an  hour  ; 
in  the  second  hour  these  would  be  multiplied  to  four; 
and  in  three  days  they  would,  if  their  surroundings  were 
ideally  favorable,  form  a  mass  which  can  scarcely  be  reck- 
oned in  numbers."  "  Fortunately  for  us,"  says  Woodhead, 
"  they  can  seldom  get  food  enough  to  carry  on  this  appalling 
rate  of  development,  and  a  great  number  die  both  for  want 
of  food  and  because  of  the  presence  of  other  conditions 
unfavorable  to  their  existence." 

Sporulation. — When  the  conditions  for  rapid  multiplica- 
tion by  fission  are  no  longer  good,  many  of  the  organisms 
guard  against  extinction  by  the  formation  of  spores  (Fig.  i). 


Bacteria  37 

Endospores,  or  spores  developed  within  the  cells,  are  gen- 
erally formed  in  the  elongate  bacteria,  —  bacillus  and  spiril- 
lum, —  but  Zopf  has  observed  similar  bodies  in  micrococci. 
Escherich  also  claims  to  have  found  undoubted  spores  in  a 
sarcina. 

Spores  may  be  either  round  or  oval.     As  a  rule,  each 

a  b  c  d  e  } 

O    o       (3^=> 


Fig.  1.  —  Diagram  illustrating  sporulation:  a,  Bacillus  inclosing  a 
small  oval  spore;  b,  drumstick  bacillus,  with  the  spore  at  the  end;  ct 
clostridium;  d,  free  spores;  e  and  /,  bacilli  escaping  from  spores. 

organism  produces  a  single  spore,  which  is  situated  either 
at  its  center  or  at  its  end.  When,  as  sometimes  happens, 
the  diameter  of  the  spore  is  greater  than  that  of  the 
bacillus,  it  causes  a  peculiar  barrel  shape  bulging  of  the 
organism,  described  as  clostridium.  When  the  distending 
spore  is  at  the  end,  a  "Trommelschlager,"  or  "drum- 
stick," is  formed.  End-spores  are  almost  characteristic 
of  anaerobic  bacilli.  When  the  formation  of  a  spore  is 
about  to  commence,  a  small  bright  point  appears  in  the 
cytoplasm,  and  increases  in  size  until  its  diameter  is  nearly 
or  quite  as  great  as  that  of  the  bacterium.  A  dark,  highly 
refracting  capsule  is  finally  formed  about  it.  As  soon  as 
the  spore  arrives  at  perfection  the  bacterium  seems  to  die, 
as  if  its  vitality  were  exhausted. 

The  spores  differ  from  the  bacteria  in  that  their  capsules 
prevent  evaporation  and  enable  them  to  withstand  drying 
and  the  application  of  a  considerable  degree  of  heat.  Very 
few  adult  bacteria  are  able  to  resist  temperatures  above 
70°  C.  Spores  are,  however,  uninjured  by  such  temper- 
atures, and  can  even  successfully  resist  the  temperature  of 
boiling  water  (100°  C.)  for  a  short  time.  The  extreme 
desiccation  caused  by  a  protracted  exposure  to  a  dry  tem- 
perature of  150°  C.  will  invariably  destroy  them,  as  will  also 
steam  under  pressure.  Not  only  can  the  spores  successfully 
resist  a  considerable  degree  of  heat,  but  they  are  also  un- 
affected by  cold  of  almost  any  intensity.  Von  Szekely*  found 
anthrax  spores  capable  of  germination  after  eighteen  years 

*  "Zeitschr.  fur  Hygiene,"  1903,  xuv,  3. 


38    Structure  and  Classification  of  Micro-organisms 

and  six  months  in  some  dried-up  old  gelatin  cultures  found 
in  his  laboratory. 

Arthrospores. — The  formation  of  arthrospores  is  less  clear, 
and  seems  to  be  the  conversion  of  the  entire  organism  into  a 
spore  or  permanent  form.  Arthrospores  have  been  observed 
particularly  among  the  micrococci,  where  certain  individuals 
become  enlarged  beyond  the  normal,  and  surrounded  by  a 
capsule. 

Though  the  cell-wall  of  the  adult  bacterium  is  easily  pen- 
etrated by  solutions  of  the  anilin  dyes,  it  is  difficult  to  stain 
spores,  which  are  distinctly  more  resistant  to  the  action  of 
chemic  agents  than  the  bacteria  themselves. 

Germination  of  Spores. — When  a  spore  is  about  to  germi- 
nate, the  contents,  which  have  been  clear  and  transparent, 
become  granular,  the  body  increases  slightly  in  size,  the 
capsule  becomes  less  distinct,  and  in  the  course  of  time 
splits  open  to  allow  the  escape  of  a  young  organism.  The 
direction  in  which  the  capsule  ruptures  varies  in  different 
species.  Bacillus  subtilis  escapes  from  the  side  of  the  spore; 
Bacillus  anthracis  from  the  end.  This  difference  can  be 
made  use  of  as  an  aid  in  differentiating  otherwise  similar 
organisms. 

So  soon  as  the  young  bacillus  escapes  it  begins  to  in- 
crease in  size,  develops  a  characteristic  capsule,  and  presently 
begins  the  propagation  of  its  species  by  fission. 

Morphology. — The  three  principal  forms  of  bacteria  are 
spheres  (cocci),  rods  (bacilli),  and  screws  (spirilla). 

Cocci. — The  spheric  bacteria,  from  a  fancied  resemblance  to 
little  berries,  are  called  cocci  or  micrococci.  When  they  divide, 
and  the  resulting  organisms  remain  attached  to  one  another, 
a  diplococcus  is  produced.  Diplococci  may  consist  of  two  at- 
tached spheres,  though  each  half  commonly  shows  flattening 
of  the  contiguous  surfaces.  In  a  few  cases,  as  the  gonococcus, 
the  approximated  surfaces  may  be  slightly  concave,  causing 
the  organism  to  resemble  the  German  biscuit  called  a  "Sem- 
mel."  When  a  second  binary  division  occurs,  and  four  result- 
ing individuals  remain  attached  to  one  another,  without  dis- 
turbing the  arrangement  of  the  first  two,  a  tetrad,  or  tetracoc- 
cus,  is  formed.  To  the  entire  groups  of  cocci  dividing  in  two 
directions  of  space  so  as  to  produce  fours,  eights,  twelves, 
etc.,  on  the  same  plane,  the  name  merismopedia  has  been 
given.  Migula  uses  the  term  micrococcus  for  the  unflagel- 
lated  tetrads,  and  planococcus  for  the  flagellated  forms. 


Bacteria  39 

If  division  take  place  in  three  directions  of  space,  so  as 
to  produce  a  cubic  "  package"  of  cocci,  the  resulting  aggre- 
gation is  described  as  a  sarcina.  This  form  resembles  a 
dice  or  a  miniature  bale  of  cotton.  Few  sarcinae  have 
flagella,  similar  flagellated  organisms  being  called  by  Migula 
planosarcina. 

If  division  always  take  place  in  the  same  direction,  so 
that  the  cocci  remain  attached  to  one  another  like  a  string 
of  beads,  the  organism  is  described  as  a  streptococcus. 

Cocci  commonly  occur  in  irregular  groups  having  a  fan- 
cied resemblance  to  bunches  of  grapes.  Such  are  called* 

/ 


<D  © 

©  O 


Fig.  2. — Diagram  illustrating  the  morphology  of  the  cocci :  a,  Coccus 
or  micrococcus;  b,  diplococcus;  c,  d,  streptococci;  e,  f,  tetracocci  or 
merismopedia ;  g,  h,  modes  of  division  of  cocci;  i,  sarcina;  /,  coccus 
with  flagella ;  k,  staphylococci. 

staphylococci,  and  most  organisms  not  finding  a  place  in  the 
varieties  already  described  are  so  classed. 

Cocci  associated  in  globular  or  lobulated  clusters  incased 
in  a  resisting  gelatinous,  homogeneous  mass,  have  been 
described  by  Billroth  as  ascococcus,  Cocci,  solitary  or  in 
chains,  surrounded  by  an  incasement  of  almost  cartilaginous 
consistence,  have  been  called  leuconostoc. 

Bacilli. — Better  known,  if  not  more  important,  bacteria 
consist  of  elongate  or  "  rod-shaped  forms,"  and  bear  the 
name  bacillus  (a  rod) .  These  present  considerable  variation 
of  form.  Some  are  ellipsoid,  some  long  and  slender.  Some 
have  rounded  ends,  as  Bacillus  subtilis;  others  have  square 
ends,  as  B.  anthracis.  Some  are  large,  some  exceedingly 
small.  Some  always  occur  singly,  never  uniting  to  form 
threads  or  chains;  others  are  nearly  always  so  conjoined. 

The  bacilli  divide  by  transverse  fission  only,  so  that  the 
only  peculiarity  of  arrangement  is  the  formation  of  threads 
or  chains.  In  the  older  writings,  short,  stout  bacilli  were 


40    Structure  and  Classification  of  Micro-organisms 

described  under  the  generic  term  bacterium.  Migula  now 
employs  the  term  to  include  only  bacillary  forms  without 
flagella.  A  pseudomonas  is  a  bacillary  form  with  polar 
flagella.  Some  of  the  flexile  bacilli  have  sinuous  move- 
ments resembling  the  swimming  of  a  snake  or  an  eel,  and 


Fig.  3. — Diagram  illustrating  the  morphology  of  the  bacilli:  a,  6, 
c,  Various  forms  of  bacilli ;  d,  e,  bacilli  with  flagella ;  /,  chain  of  bacilli, 
individuals  distinct ;  g,  chain  of  bacilli,  individuals  not  separated. 

are  sometimes  described  as  vibrio;  but  this  name  also  has 
passed  into  disuse,  except  in  France,  where  spiral  organisms 
are  so  called. 

Spirilla. — If  a  rod-shaped  bacterium  is  spirally  twisted 
and  resembles  a  corkscrew,  it  is  called  spirillum.  The  rigid 
forms  without  flagella  are  known  as  spirosoma;  rigid  forms 
with  flagella,  spirilla  and  microspira. 

A  spiral  organism  of  ribbon  shape  is  called  spiromonas, 
while  a  similar  organism  of  spindle  shape  is  called  a  spiru- 
lina.  One  species  of  spiral  bacteria  in  whose  cytoplasm 


Fig.  4. — Diagram  illustrating  the  morphology  of  the  spirilla:    a,  b,  c, 

Spirilla. 

sulphur  granules  have  been  detected  has  been  called  ophi- 
diomonas. 

Spiral  organisms  with  undulating  membranes  are  known  as 
spiroch&ta,  but  these  and  the  similar  genus  treponema  are 
now  regarded  as  more  correctly  placed  among  the  protozoan 
organisms. 


The  Higher  Bacteria 


THE  HIGHER  BACTERIA. 

The  Higher  Bacteria  form  a  group  intermediate  between 
the  Schizomycetes,  or  true  bacteria,  and  the  Hyphomycetes, 
or  molds.  In  the  classification  of  Migula  and  Chester 
they  include  the  Mycobacteriaceae  and  the  Chlamydobac- 
teriaceae.  Some,  like  Petruschky,  believe  them  to  be  more 
closely  related  to  the  true  molds  than  to  the  bacteria. 
They  are  characterized  by  filamentous  forms  with  real  or 
apparent  branchings.  The  filaments  are  usually  regularly 
divided  transversely,  so  as  to  appear  as  if  composed  of  bacilli. 


Fig.  5. — Cladothrix,  showing  false  branching.    (From  Hiss  and  Zinsser, 
"  Text-Book  of  Bacteriology,"  D.  Appleton  &  Co.,  publishers.) 

The  free  ends  only  seem  to  be  endowed  with  reproductive 
functions,  and  develop  peculiar  elements  that  differentiate 
the  higher  from  the  other  bacteria  whose  cells  are  all  equally 
free  and  independent. 

Leptothrix. — These  comprise  long  threads  which  do  not 
branch.  They  are  not  always  easily  separated  from  chains 
of  bacilli.  They  rarely  appear  to  play  a  pathogenic  role, 
though  those  inhabiting  the  mouth  occasionally  secure  a 
foothold  upon  the  edges  of  the  tonsillar  crypts,  where  they 
grow,  with  the  formation  of  persistent  white  patches.  This 
form  of  leptothrix  mycosis  is  chronic  and  difficult  to  treat. 


42     Structure  and  Classification  of  Micro-organisms 

The  leptothrix  is  a  very  difficult  organism  to  secure  in  culture. 
The  attempts  of  Vignal*  and  of  Arustamoff  f  were  successful, 
but  upon  the  usual  culture-media  the  organisms  grew  very 
sparingly. 

Cladothrix. — These  also  produce  long  thread-like  filaments, 
but  they  occasionally  show  what  is  described  as  false  branch- 
ing; that  is,  branches  seem  to  originate  from  the  threads, 
but  no  distinct  connection  between  the  thread  and  the  ap- 
parent branch  obtains.  None  of  the  cladothrices  is  known 


Fig.  6. — Streptothrix  enteola.      Film  preparation  from   peptone-beef- 
broth  culture,  fourteen  days  at  37°  C.  X  1000.     (Foulerton.) 


to  be  pathogenic.  They  are  frequently  organisms  of  the  at- 
mospheric dust,  and  not  infrequently  appear  as  "weeds" 
in  culture-media  The  colonies  grow  to  about  a  centimeter  in 
diameter,  are  usually  white  in  color,  irregularly  rounded, 
sharp  at  the  edges,  more  or  less  concentric,  dry  and  pow- 
dery (not  velvety)  or  scaly  on  the  surface.  They  commonly 
liquefy  gelatin  and  blood-serum. 

Streptothrix. — These  organisms  certainly  branch.  They 
also  form  endospores.  Many  of  them  can  be  cultivated. 
Not  a  few  are  found  under  circumstances  suggesting  patho- 

*  "Annales  de  physiologic,"  1886. 

*  Kolle    and    Wassermann,    "Handbuch    der    Pathogenen    Mikro- 
organismen,"  1903,  n,  p.  851;  Wratsch,i889. 


The  Yeasts,  or  Blastomycetes  43 

genie  action.  For  a  long  time  there  has  been  a  disposition 
to  regard  Bacillus  tuberculosis  as  a  form  of  strep  to  thrix,  since 
old  cultures  show  branching  involution  forms.  The  old 
genus  actinomyces  is  also  included  by  a  number  of  writers 
among  the  streptothrices,  so  that  the  Actinomyces  bovis  of 
Bollinger  is  called  Strep  to  thrix  actinomyces,  the  Actinomyces 
madurae,  Streptothrix  madurae,  and  the  organism  found  by 
Nocard  in  the  disease  known  as  "  farcin  du  bauf,"  Strepto- 
thrix farcinica.  There  seems,  however,  no  adequate  ground 
for  this  arrangement,  and  the  old  genus  Actinomyces  should 
be  kept.  Bppinger,  found  a  streptothrix  in  the  pus  of  a 
cerebral  abscess,  and  Petruschky,  Berestneff,  Flexner, 
Norris,  and  Larkin  have  found  streptothrices  in  cases  of 
pulmonary  disease  simulating  tuberculosis.  The  organisms 
described  by  these  writers  were  not  identical,  so  that  there  are 
probably  several  different  species.  They  usually  grow  well 
upon  ordinary  media  and  upon  solid  media  form  whitish, 
glistening,  well-circumscribed  colonies  attaining  a  diameter 
of  several  millimeters.  As  they  grow  old  they  turn  yellowish 
or  brownish.  They  liquefy  gelatin.  Some  of  the  cultures 
were  not  harmful  to  the  laboratory  animals,  others  caused 
suppuration. 

Actinomyces. — The  chief  characterization  of  the  organisms 
of  this  group  is  a  clavate  expansion  of  the  terminal  ends  of 
radiating  filaments.  These  are  seen  in  sections  of  diseased 
tissues  containing  the  organisms,  but  rarely  are  well  shown  in 
the  artificial  cultures.  For  further  particulars  of  these  or- 
ganisms see  Actinomyces  bovis,  etc. 


THE  YEASTS,  OR  BLASTOMYCETES. 

The  organisms  of  this  group  are  sharply  separated  from  the 
bacteria  by  their  larger  size,  elliptic  form,  and  by  multipli- 
cation by  gemmation  or  budding. 

Each  organism  is  surrounded  by  a  sharply  defined, 
doubly  contoured,  highly  refracting,  transparent  cellulose 
envelope.  Commonly  each  cell  contains  one  or  more  distinct 
vacuoles.  When  multiplication  is  in  progress,  smaller  and 
larger  buds  are  formed. 

The  yeasts,  of  which  Saccharomyces  cerevisiae  may  be 
taken  as  the  type,  are  active  fermentative  organisms,  quickly 
splitting  the  sugars  into  CO2  and  alcohol,  and  are  largely  cul- 


44    Structure  and  Classification  of  Micro-organisms 

tivated  and  used  in  the  manufacture  of  fermented  liquors 
and  bread.  They  grow  well  in  fermentable  culture-media 
and  most  of  them  also  grow  upon  the  ordinary  laboratory 
culture-media.  Many  varieties,  some  of  which  produce  red 
or  black  pigment,  some  non-chromogenic,  are  known.  They 


Fig.  7. — Blastomycetes  dermatitidis.  Budding  forms  and  mycelial 
growth  from  glucose  agar  (Irons  and  Graham,  in  "Journal  of  Infectious 
Diseases"). 

play  very  little  part  in  the  pathogenic  processes.  Burse  has 
observed  a  case  of  generalized  fatal  infection  caused  by  an 
yeast  that  he  calls  Saccharomyces  hominis.  Gilchrist, 
Curtis,  Ophuls,  and  others  have  seen  localized  human  infec- 
tions by  blastomycetes.  (See  Blastomycetic  dermatitis.) 


THE  OIDIA. 

These  organisms  seem  to  occupy  a  place  intermediate  be- 
tween the  yeasts  and  the  molds — the  blastomycetes  and  the 
hyphomycetes.  In  certain  stages  they  appear  as  oval  cells 
which  multiply  by  gemmation,  but  instead  of  becoming  sep- 
arated, hang  together.  At  a  later  stage  of  development  they 
grow  into  long  filamentous  formations  suggesting  the  mycelia 
of  molds,  but  being  less  regular.  Certain  cells  also  develop 
as  reproductive  organs. 


The  Oidia 


45 


They  are  common  micro-organisms  of  the  air  and  appear  as 
frequent  causes  of  contamination  in  culture-media,  upon  all 


Fig.  8. — Oidium,  showing  the  various  vegetative  and  reproductive  ele- 
ments.    X  350  (Grawitz). 

forms  of  which  they  grow  readily,  producing  liquefaction 
where  possible.  They  engage  in  but  few  pathogenic  processes, 
the  most  familiar  being  that  brought  about  by  Oidium  albi- 


Fig.  9. — Oidium  (Kolle  and  Wassermann). 

cans,  which  causes  the  common  disease  of  childhood  known  as 
thrush  (q.  v.). 


46    Structure  and  Classification  of  Micro-organisms 

THE  MOLDS. 

In  this  group  it  is  customary  to  place  a  miscellaneous 
collection  of  organisms  having  in  common  the  formation  of 
a  well-marked  mycelium,  but  being  so  diversified  in  other  re- 
spects as  to  place  them  in  widely  separated  groups  in  the  sys- 
tematic arrangement  of  the  fungi.  Some  are  correctly 
placed  among  the  "  Imperfect  fungi,"  some  among  the  Asco- 
mycetes,  and  some  among  the  Phy corny cetes.  They  are  all 
active  enzymic  agents  and  produce  fermentative  and  putre- 
factive changes. 


Fig.  10. — Mucor  mucedo:  i,  A  sporangium  in  optical  longitudinal 
section;  c,  columella;  m,  wall  of  sporangium;  sp,  spores;  2,  a  ruptured 
sporangium  with  only  the  columella  (c)  and  a  small  portion  of  the  wall 
(m)  remaining;  3,  two  smaller  sporangia  with  only  a  few  spores  and  no 
columella;  4,  germinating  spores;  5,  ruptured  sporangium  of  Mucor 
mucilaginus  with  deliquescing  wall  (m)  and  swollen  interstitial  substance 
(z);  sp,  spores  (after  Brefeld). 

1.  Ackorion. — The  organisms  of  this  genus  are  character- 
ized by  a  more  or  less  branched  hypha,  3  to  5  ^  in  diameter, 
which  break  up  after  a  time  into  rounded  or  cuboidal  spores. 
The  Achorion  schonleini  is  highly  pathogenic  and  will  be 
described  in  the  section  upon  Favus. 

2.  Tricophyton  and  Microsporon. — These  names  are  applied 
somewhat  loosely  to  organisms  affecting  skin  and  hair  fol- 
licles of  men  and  animals.     They  form  tangled  slender  my- 
celia   with   many   spores   of   varying   size.     They   occasion 


The  Molds 


47 


"ringworm,"  barber's  itch,  pityriasis,  and  tinea.  Further 
description  of  the  organisms  will  be  found  in  the  section  upon 
Ringworm. 

3.  Mucor. — The  mucors,  or  "black  molds,"  belong  to  the 
phy corny cetes.  They  form  a  thick,  tangled  mycelium,  in 
and  above  which  the  rounded  black  sporongia  can  be  seen 
with  the  naked  eye.  The  mycelium  becomes  divided  at  the 
time  of  reproduction.  Multiplication  takes  place  asexually 
through  conidia-spores  which  develop  within  sporangia,  and 
sexually  by  the  conjugation  of  specialized  terminal  septate 


Fig.  1 1 . — Mucor  mucedo.    Single-celled  mycelium  with  three  hyphae  and 
one  developed  sporangium  (after  Kny,  from  Tavel). 

branches  of  the  mycelium,  which  conjugate  with  similar 
cells,  belonging  to  other  colonies,  to  form  zygospores. 

The  sporangia  form  upon  the  ends  of  aerial  hypha  and  con- 
sist of  a  smooth  spherical  capsule  within  which  the  spores 
develop,  to  become  liberated  only  when  the  membrane  rup- 
tures. The  colonies,  each  of  which  is  unisexual,  may  be 
described  as  +  and  — .  Colonies  of  the  +  type  will  not 
conjugate;  colonies  of  the  —  type  will  not  conjugate,  but 
when  terminal  filaments  of  +  and  —  come  together,  conjuga- 
tion occurs  and  zygospore  formation  takes  place. 

Mucors  are  not  infrequent  organisms  of  the  atmosphere 
and  occasionally  appear  as  contaminations  upon  solid  culture- 
media.  About  130  species  are  known.  Of  these,  Mucor 
corymbifer,  Mucor  rhizopodiformis,  Mucor  ramosus,  Mucor 


48    Structure  and  Classification  of  Micro-organisms 

pusillus,  Mucor  septatus,  and  Mucor  conoides  are  said  by 
Plaut*  to  be  pathogenic  when  introduced  into  laboratory 
animals.  Mucor  corymbifer  has  been  known  to  produce 
inflammation  of  the  external  auditory  meatus  in  man.f 
General  mucor  mycosis  in  man  has  also  been  observed  by 
Paltauf  J  to  result  from  the  presence  of  the  same  organism. 


Fig.  12. — Mucor  mucedo.  Different  stages  in  the  formation  and 
germination  of  the  zygospore:  i,  Two  conjugating  branches  in  contact; 
2,  septation  of  the  conjugating  cells  (a)  from  the  suspensors  (&) ;  3,  more 
advanced  stage  in  the  development  of  the  conjugating  cells  (a) ;  4,  ripe 
zygospore  (6)  between  the  suspensors  (a);  5,  germinating  zygospore 
with  a  germ-tube  bearing  a  sporangium  (after  Brefeld). 


4.  Aspergillus  and  Eurotium. — The  organisms  of  this  genus 
are  included  among  the  Ascomycetes.  They  are  common 
organisms  of  the  air  and  frequent  contaminations  of  the  solid 
culture-media.  To  secure  them  an  agar-agar  plate  can  be 
exposed  to  the  atmosphere  of  the  laboratory  for  a  short  time, 
then  covered  and  stood  aside  for  a  day  or  two,  when  tangled 
mycelial  growths  with  rapidly  spreading  hyphae  will  usually 
be  discovered.  The  recognition  is  easily  made  when  the 
sporangia  appear.  These  are  well  shown  in  the  accompany- 
ing illustration.  The  mycelium  is  divided  into  many  cells. 

*  Kolle  and  Wassermann,  "Die  Pathogenen  Mikroorganismen," 
1903,  i,  552. 

t  Htickel-Losch  in  Fliigge,  "Die  Mikroorganismen."  |  Ibid. 


The  Molds 


49 


Reproduction  is  asexual  and  takes  place  through  conidia 
spores.  The  fruit  hyphae,  which  are  aerial,  terminate  in 
rounded  extremities  which  are  known  as  columella,  from 
which  many  radiating  sterigmata  arise,  each  terminating  in  a 
series  of  rounded  spores.  A  sexual  form  of  reproduction  also 
takes  place  through  the  production  of  ascospores.  Many 
species  are  known,  only  a  few  of  which  are  pathogenic. 


Fig.  13. — Aspergillus  glaucus:  A,  A  portion  of  the  mycelium  m,  with 
a  conidiaphore  c,  and  a  young  perithrecium  F,  magnified  190  diameters; 
B  and  B',  conidiaphore  with  conidia;  B,  individual  sterigma  greatly 
magnified;  C,  early  stage  of  the  development  of  the  fructifying  organ; 
D  young  perithrecium  in  longitudinal  section;  w,  the  future  wall  of 
the  contents;  as,  the  screw,  magnified  250  diameters;  E,  an  ascus  with 
spores  from  a  perithrecium,  magnified  600  diameters  (duBary). 

Aspergillus  malignum  has  been  found  by  von  Lindt  in  the 
auditory  meatus  of  man. 

Aspergillus  nidulans  occasionally  infects  cattle.  It  is 
pathogenic  for  laboratory  animals,  usually  causing  death  in 
sixty  hours.  The  kidneys  are  found  enlarged  to  twice  their 
normal  size,  and  show  small  whitish  dots  and  stripes  of  cell 
infiltration  containing  the  fungi.  The  heart  muscle,  dia- 
phragm, and  spleen  may  also  be  involved.  The  liver  usually 
escapes.  It  takes  a  large  number  of  spores  to  infect. 

Aspergillus  fumigatus. — This  is  a  widespread  and  not  in- 
frequently pathogenic  form.  Its  most  common  lesion  is  a 


50    Structure  and  Classification  of  Micro-organisms 

pneumomycosis,  in  which  the  lung  is  riddled  with  small 
inflammatory  necrotic  and  cavernous  areas  containing  the 
molds.  The  same  condition  has  occasionally  been  observed 
in  human  beings,  Sticker  having  collected  39  cases.* 

Leber  and  others  have  observed  keratitis  following  corneal 
infection  by  this  organism. 

Aspergillus  flavus  is  also  pathogenic. 

Aspergillus  subfuscus  is  also  pathogenic  and  highly  virulent. 

Aspergillus  niger. — Pathogenic  and  found  at  times  in 
inflammation  of  the  external  auditory  meatus. 

5.  Penicillium. — These  are  common  green  molds,  widely  dis- 
seminated throughout  the  atmosphere  and  frequent  sources 
of  contamination  of  the  culture-media  in  the  laboratory. 


Fig.  14 — Penicillium  (Eyrej. 

Moist  bread  exposed  to  the  atmosphere  soon  becomes  covered 
with  them.  They  are  included  in  the  group  of  fungi  imper- 
fecti,  and  are  characterized  by  a  luxuriant  tangled  septate 
mycelium,  with  aerial  fruit  hyphae,  ending  in  conidiophores, 
each  of  which  divides  into  two  or  three  sterigmata,  the  tip  of 
which  forms  a  chain  of  rounded  spores.  The  whole  germinal 
organ  thus  comes  to  resemble  a  whisk-broom  or,  as  Hiss  de- 
scribes it,  a  skeleton  hand,  in  which  the  conidiophore  corre- 
sponds to  the  wrist;  the  sterigmata,  to  the  metacarpal  bones; 
the  chains  of  spores,  to  the  phalanges. 

None  of  the  penicillia  is  known  to  be  pathogenic  either  for 
man  or  animals. 

Penicillium  crustaceum  (glaucum)  is  the  most  common 
source  of  contamination  of  the  laboratory  media. 

Penicillium  minimum,  which  may  be  identical  with  the 
preceding,  was  once  found  in  the  human  ear  by  Sievenmann. 

*  Nothnagel's  Spezielle  Path.  u.  Therap.,  xiv,  1900. 


The  Protozoa  51 


THE  PROTOZOA. 

The  protozoa  are  unicellular  animal  organisms  as  differen- 
tiated from  the  metazoa  which  are  multicellular  animal  or- 
ganisms. The  restriction,  implied  by  the  term  unicellular  is, 
however,  too  narrow,  for  there  are  colonial  protozoa  that  con- 
sist of  many  cells,  yet  share  other  protozoan  characters. 

For  the  purposes  of  this  work,  however,  all  protozoa  are 
to  be  regarded  as  unicellular  and  the  individuals  independent 
of  one  another. 

Classification.  —  Many  schemes  have  been  devised  for 
systematically  arranging  the  protozoa,  that  which  follows 
being  an  abbreviation  of  the  standard  classification,  made  to 
correspond  with  the  requirements  of  this  work  that  deals 
only  with  the  pathogenic  forms. 

CLASSIFICATION   OF  THE   PATHOGENIC   PROTOZOA. 


Phylum   PROTOZOA  (Trpwrof  first,  Cwov  animal).      Unicellular  ani- 

mal organisms. 

Class  Rhizopoda  (pifa  root,  7rw(5of  foot).  Having  soft  plasmic 
bodies  with  or  without  external  protecting  shells.  The  con- 
tour subject  to  change  through  the  formation  of  extensions 
known  as  pseudopods.  These  may  be  blunt,  rounded,  or 
lobose,  filamentous,  or  anastomosing.  The  nutrition  is 
holozoic  or  holophytic. 
Order  GYMNAMCEBA  (yvfj.v6c  naked).  Rhizopoda  without  ex- 

ternal shells  or  coverings. 
Genus  Amoeba  (a^oifta  to  change). 
Entamoeba. 
Chlamydophrys. 
"      Leydenia. 

Class  Mastigophora  (udanyos  whips,  <t>6poc  to  bear).  Organ- 
isms of  well-defined  form,  naked  or  surrounded  by  a  well- 
defined  membrane.  Nutrition  is  holozoic,  holophytic,  para- 
sitic, or  saprophytic.  Mouth,  contractile  vesicle,  and  nucleus 
usually  present. 

Order  FLAGEXLATA  (Latin,  flagellare,  to  beat).  Small  organisms 
with  a  well-defined  mononucleate  body,  at  the  anterior  end 
or  both  ends  of  which  are  one  or  more  flagella.  Actively 
motile.  May  become  encysted.  Nutrition  is  holozoic,  holo- 
phytic, parasitic,  or  saprophytic. 
Family  CercomonidcB.  Body  pyriform,  with  several  anterior 

flagella  and  an  undulating  membrane. 
Genus  Cercomonas. 
"      Trichomonas.    . 
Monas. 
Plagiomonas. 

Family  Lambliadce.     Body  pyriform,  very  much  attenuated 
behind.     Ventral   surface   shows  a  reniform   depression, 
about  the  posterior  part  of  which  there  are  six  flagella. 
There  are  also  two  flagella  at  the  posterior  extremity. 
Genus  Lamblia  (Megastomum). 


52    Structure  and  Classification  of  Micro-organisms 

Family     Trypanosomida.     Body    delicately    fusiform.     Con- 
tains a   nucleus,  a  blepharoplast  or  centrosome,  and  an 
undulating  membrane.     A  single  wavy  flagellum  arises 
in  the  posterior  part  of  the  body  close  to  the  centrosome, 
passes  along  the  edge  of  the  undulating  membrane  to  the 
anterior  extremity,  where  it  continues  free  for  some  dis- 
tance.    Nutrition  parasitic.     Reproduces  by  division. 
Genus  Trypanosoma. 
"      Leishmania. 

Babesia. 

Family    Spiroch<ztid(B.     Organisms    very    long    and    spirally 

twisted.     Nucleus    indistinct.     Multiplication    probably 

by  longitudinal  division  only.     Nutrition  is  parasitic  or 

saprophytic. 

Genus  Spirochaeta.     Body  flattened,  with  a  very  narrow 

undulating  membrane. 

Genus  Treponema.  Body  not  flattened.  No  undulating 
membrane.  Extremities  sharp  pointed  and  terminating 
in  short  flagella. 

Class  Sporozoa  (cxopof  a  spore,  faov  an  animal).  Organisms 
unprovided  with  cilia  or  flagella  in  the  adult  stage.  Always 
endoparasites  in  the  cells,  tissues,  or  cavities  of  other  animals. 
Nutrition  is  parasitic  and  osmotic.  Reproduction  always  by 
spore-formation,  the  germs  or  sporozoites  either  being  produced 
by  the  parent  or  indirectly  from  spores,  into  which  the  parent 
divides. 
Subclass  Telosporidia.  Spore-formation  ends  the  individual  life, 

the  entire  organism  being  transformed  to  spores. 
Order  GREGARINIDA.  Possess  distinct  membrane  with  myo- 
nemes  during  adult  life;  locomotion  mainly  by  contraction. 
Young  stages  alone  (cephalonts)  are  intracellular  parasites, 
the  adults  (sporonts)  being  found  in  the  digestive  tract  or  the 
body  cavities.  Sporulation  takes  place  after  or  without 
conjugation,  but  within  a  cyst  that  is  never  formed,  while 
the  parasite  is  intracellular. 

Order  COCCIDIIDA.     Spherical  or  ovoid  in  form,   without  a 
free  and  motile  adult  stage.     Never  ameboid.     Sporula- 
tion takes  place  within  cysts  formed  while  the  organism  is 
an  intracellular  parasite. 
Genus  Coccidium. 

"      Eimeria. 

Order  H^MOSPORIDIIDA.  Sporozoa  of  small  size  living  in  the 
blood-corpuscles  or  plasma  of  vertebrates.  The  adult 
form  is  mobile  and  in  some  cases  provided  with  myonemes. 
Reproduction  by  endogenous  or  asexual  sporulation,  while 
in  the  host  or  by  exogenous  sporulation  after  conjugation. 
Genus  Plasmodium. 

Subclass  Neosporidia.  Organisms  that  form  sporocysts  through- 
out life,  the  entire  cell  not  being  used  up  in  the  formation  of 
the  spores. 

Order  SARCOSPORIDIA.     The  initial  stage  of  the  life  history  is 
passed    in    the    muscle    cells    of    vertebrates.     Form    is 
elongate,  tubular,  oval,  or  even  spherical.     Cysts  have  a 
double  membrane,  in  which  reniform  or  falciform  sporo- 
zoites are  formed. 
Genus  Sarcocystis. 
"      Miescheria. 
"      Balbiania. 


The  Protozoa  53 

Subclass   Haplosporidia.       Spores    provided   with   large   round 

nuclei.     No  polar  capsules. 
Genus  Rhinosporidium. 

Class  Infusoria  (Latin,  injusus,  to  pour  into.  The  organisms  were 
given  this  name  because  they  were  first  found  in  infusions 
exposed  to  the  air).  Protozoa  in  which  the  motor  apparatus 
is  in  the  form  of  cilia,  either  simple  or  united  into  membranes, 
membranelles,  or  cirri.  The  cilia  may  be  permanent  or 
limited  to  the  embryonic  stages.  There  are  two  kinds  of 
nuclei,  macronucleus  and  micronucleus.  Reproduction  is 
effected  by  simple  transverse  division  or  by  budding.  Nutri- 
tion is  holozoic  or  parasitic. 

Subclass  Ciliata.  Mouth  and  anus  usually  present.  The 
contractile  vacuole  often  connected  with  a  complicated  sys- 
tem of  canals. 

Order  HOLOTRICHIDA.     The  cilia  are  similar  and  distributed 
all  over   the  body,  with  a  tendency  to  lengthen  at    the 
mouth.     Trichocysts  are  always  present,  either  over  the 
whole  body  or  in  special  regions. 
Genus  Colpoda. 
"      Chilodon. 

Order    HETEROTRICHIDA.     Organisms    possessing    a    uniform 
covering  of  cilia  over  the  entire  body,  and  an  adoral  zone 
consisting  of  short  cilia  fused  together  into  membranelles. 
Suborder  Polytrichina.     Uniform  covering  of  cilia. 

Family  Bursarida.  The  body  is  usually  short  and  pocket- 
like,  but  may  be  elongated.  The  chief  characteristic 
is  the  peristome,  which  is  not  a  furrow,  but  a  broad 
triangular  area  deeply  insunk,  and  ending  in  a  point 
at  the  mouth.  The  adoral  zone  is  usually  confined 
to  the  left  peristome  edge  or  it  may  cross  over  to  the 
right  anterior  edge. 
Genus  Balantidium. 

Structure. — From  the  table  it  will  at  once  be  evident  that 
the  protozoa  form  an  extremely  varied  group,  and  that  no 
kind  of  descriptive  treatment  can  be  looked  upon  as  adequate 
that  does  not  consider  individuals. 

Cytoplasm. — In  some  of  the  smaller  protozoa,  and  in  certain 
stages  of  others,  the  cytoplasm  appears  almost  hyaline  and 
structureless.  In  most  cases,  however,  it  appears  granular, 
and  in  the  larger  organisms,  such  as  ameba,  it  presents  the 
appearance  which  some  described  as  granular,  others,  as 
frothy.  The  accepted  theory  of  structure  teaches  that  the 
protoplasm  is  honeycombed  or  frothy,  and  that  it  is  filled 
with  endless  chambers  in  which  its  enzymes  and  other 
active  substances,  etc.,  are  stored  up  and  its  functions  car- 
ried on. 

In  addition  to  these  chambers,  which  are  minute  and  of 
uniform  size,  there  are  larger  spaces  called  vacuoles,  some 
of  which  are  the  result  of  temporary  conditions — accumu- 
lations of  digested  but  not  yet  assimilated  food,  etc.;  but 


54    Structure  and  Classification  of  Micro-organisms 

others,  seen  in  ameba  and  in  the  ciliata,  are  large,  per- 
manent, and  characterized  by  rhythmical  contractions 
through  which  they  disappear  from  one  part  of  the  body  sub- 
stance to  appear  in  another.  These  are  known  as  "  con- 
tractile vacuoles,"  and  are  supposed  to  subserve  the  useful 
purpose  of  assisting  in  maintaining  cytoplasttiic  currents  and 
so  distributing  the  nourishing  juices. 

The  cytoplasm  also  contains  remnants  of  undigested  or 
indigestible  foods  which  constitute  the  paraplasm  or  deutero- 
plasm.  In  a  few  cases  granules  of  chlorophyl  are  also  to 


Fig.  15. — Internal  parasites:  A,  Amoeba  coli,  Losch;  B,  Monocystis 
agilis,  I^euck.,  a  gregarine;  C,  Megastoma  entericum,  Grassi,  a"  flagel- 
late; D,  Balantidium  coli,  Ehr.,  a  ciliate. 

be  found   in  organisms   otherwise   resembling  animals  too 
closely  to  be  confused  with  plants. 

The  cytoplasm  may  be  soft  and  uniform  in  quality,  or 
there  may  be  a  surface  differentiation  into  ectosarc,  or  body 
covering,  and  endosarc,  body  substance.  In  the  rhizopoda 
there  is  little  difference  between  the  two,  though  certain 
fresh-water  ameba  cover  themselves  with  minute  grains  of 
mineral  substance,  but  in  most  of  the  mastigophora  and  in- 
fusoria corticata  the  ectosarc  is  characterized  by  a  peculiar 
rigidity  that  gives  the  animal  a  definite  and  permanent  form. 
From  the  surface  covering  or  ectosarc  coarse  threads  or  fine 
hair-like  appendages — flagella  and  cilia — often  project.  In 
many  of  the  infusoria  the  ectosarc  contains  trichocysts  from 
which  nettling  or  stinging  threads  are  thrown  out  when  the 
organisms  are  irritated. 


The  Protozoa  55 

The  body  substance  may  show  no  morphologic  differen- 
tiation in  rhizopoda,  but  in  the  corticata  there  may  not  only 
be  a  permanent  form,  but  there  may  be  adaptations,  such  as  an 
oral  aperture,  sometimes  infundibular  in  shape  and  communi- 
cating with  the  soft  endosarc  through  a  blind  tube.  An 
anal  aperture  may  also  be  present. 

In  the  higher  infusoria  the  ectosarc  may  also  be  continued 
posteriorly  to  form  a  stalk,  by  which  the  organism  attaches 
itself  ( Vorticella) .  Such  stalks  are  contractile. 

Nucleus. — In  certain  protozoa  of  very  simple  and  indefinite 
structure — spirochaeta  and  treponema — no  distinct  well- 
contoured  nucleus  can  be  observed. 

In  the  rhizopoda  the  nucleus  is  a  distinct  organ  surrounded 
by  a  nuclear  membrane  and  containing  the  usual  chromatin 
and  linin. 

The  greater  number  of  mastigophora  possess  two  distinct 
bodies,  either  a  nucleus  and  a  centrosome  or  a  major  and 
minor  nucleus.  This  is  well  shown  in  trypanosoma. 

The  infusoria  vary  greatly  in  the  character  of  the  nuclei. 
As  a  rule,  there  are  two  indefinite  nuclei,  the  macronucleus 
and  the  micronucleus.  Both  seem  to  be  essential  organs,  and 
in  the  phenomena  supervening  upon  conjugation  both  par- 
ticipate. The  nuclei  of  the  protozoa  are,  therefore,  extremely 
diversified,  and  vary  from  the  most  simple  collections  of 
granules  of  nuclear  substance  to  large  well-formed  fantastic- 
ally shaped  composite  organs. 

Movement. — Some  kind  of  movement  is  to  be  observed  at 
some  period  in  the  life  of  almost  every  protozoan. 

In  rhizopoda  with  the  soft  ectosarc  the  movement  consists 
of  flowing  currents  by  which  lobose  projections  of  the  body 
substance  appear  now  here,  now  there,  in  the  form  of  pseudo- 
podia,  or  else  a  continuous  flowing,  by  which  the  upper  surface 
continually  coming  forward  in  a  thin  layer  coincides  with  the 
progress  of  the  animal,  which  continually  rolls  over  and  over 
as  it  were. 

In  mastigophora  the  movement  of  the  more  rigid  bodies 
is  effected  through  presence  of  longer  or  shorter  flexile  or 
rigid  coarse  threads  or  "  whips."  These  usually  project  an- 
teriorly— trypanosoma — and  by  means  of  a  spiral  move- 
ment draw  the  cell  along  with  a  propeller-like  action;  sym- 
metrically arranged  flagella  may  operate  more  like  oars. 

The  sporozoa  usually  manifest  very  little  movement,  yet 
their  sporozoites  are  motile,  and  the  spermatozoits  are  also 
motile  and  commonly  flagellated. 


56    Structure  and  Classification  of  Micro-organisms 

The  infusoria  are  actively  motile  through  abundant  fine 
hair-like  formations  known  as  cilia.  These,  multitudinous 
as  they  are,  vibrate  synchronously  with  an  oar-like  move- 
ment, propelling  the  organisms  forward  or  backward  or  mak- 
ing them  revolve  with  great  rapidity.  Independent  cilia 
not  infrequently  encircle  the  oral  aperture,  causing  a  vortex, 
in  which  the  minute  structures  upon  which  the  creatures 
feed  are  caught  and  carried  into  the  body. 

Size. — The  protozoa  show  very  great  variation  in  size. 
Some  of  the  sporozoa  form  minute  parasites  of  the  red  blood- 
corpuscles  or  other  cells  of  the  vertebrates.  The  treponema 
is  so  small  that  it  can  slowly  find  its  way  through  the  pores  of 
a  Berkefeld  filter. 

On  the  other  hand,  the  sarcoporidium  is  so  large  that  one 
of  its  cysts,  composed  of  a  single  organism,  can  be  seen  with 
the  naked  eye.  Certain  protozoa  that  play  no  part  in  morbid 
processes — myxosporidia — and  so  do  not  come  within  the 
scope  of  this  work,  may  be  several  centimeters  in  diameter. 

Reproduction. — The  reproduction  of  the  protozoa  takes 
place  both  asexually  and  sexually.  It  may  be  that  there  are 
no  strictly  asexual  protozoa,  nearly  all  forms  having  been 
shown  upon  intimate  acquaintance  to  be  subject  to  occasional 
conjugation.  Conjugation  may  result  in  the  loss  of  individual 
identity  or  the  conjugated  individuals  may  again  separate. 

Whether  the  reproduction  takes  place  asexually  without 
conjugation  or  sexually  after  conjugation,  it  always  occurs  by 
division,  'which  may  be  simple  and  binary  or  complex  and 
multiple. 

Wherever  a  distinct  nucleus  can  be  found,  the  multipli- 
cation of  the  protozoa  is  preceded  by  some  kind  of  mitotic 
change.  The  more  complex  the  structure  of  the  nucleus,  the 
more  complicated  and  perfect  the  mitosis. 

The  elongate  protozoa  divide  lengthwise,  which  is  some- 
times contrary  to  expectation,  as  in  the  cases  of  treponema 
and  spirochaeta. 

The  multitudinous  sporozoi'tes  into  which  the  zygotes  of 
the  sporozoa  divide  are  commonly  the  result  of  anterior  divi- 
sion into  intermediate  bodies  known  as  oocysts,  ookinetes, 
sporocysts,  etc.  The  nuclear  substance  is  first  divided  so  as 
to  be  uniformly  distributed  among  these,  then  further  divided 
so  that  some  of  it  reaches  each  sporozoite. 

In  the  process  of  sporulation  the  entire  parent  may  be 
used  up,  as  in  the  coccidium  and  plasmodium,  or  the  parent 


The  Protozoa  57 

may  continue  to  live  and  later  form  additional  sporozoites, 
as  in  sarcocystis. 

Encystment. — Nearly  all  of  the  protozoa  are  capable  at 
times  of  encysting  themselves,  i.  e.,  surrounding  themselves 
with  dense  capsules  by  which  life  may  be  preserved  for  some 
time  amid  such  unfavorable  surroundings  as  excessive  cold, 
excessive  dry  ness,  and  absence  of  food.  Sometimes  the 
encysted  stage  is  the  spore  stage  (coccidium),  sometimes  it 
is  the  adult  stage  (ameba).  Under  these  circumstances  we 
find  an  analogy  with  the  sporulation  of  the  bacteria  which  is 
not  for  purposes  of  multiplication,  but  for  self-preservation. 
The  encysted  protozoa  are  less  hardy,  however,  than  the 
bacterial  and  other  plant  spores,  and  succumb  to  compara- 
tively slight  elevations  of  temperature. 


CHAPTER   II. 

BIOLOGY  OF  MICRO-ORGANISMS. 

THE  distribution  of  micro-organisms  is  well-nigh  universal. 
They  and  their  spores  pervade  the  atmosphere  we  breathe,  the 
water  we  drink,  the  food  we  eat,  and  luxuriate  in  the  soil 
beneath  our  feet. 

They  are  not,  however,  ubiquitous,  but  correspond  in  dis- 
tribution with  that  of  the  matter  upon  which  they  live 
and  the  conditions  they  can  endure.  Tyndall*  found  the 
atmosphere  of  high  Alpine  altitudes  free  from  them,  and 
likewise  that  the  glacier  ice  contained  none;  but  wherever 
man,  animals,  or  even  plants  live,  die,  and  decompose,  they 
are  sure  to  be  present. 

Their  presence  in  the  air  generally  depends  upon  their 
previous  existence  in  the  soil,  its  pulverization,  and  dis- 
tribution by  currents  of  the  atmosphere.  Koch  has  shown 
that  the  upper  stratum  of  the  soil  is  exceedingly  rich  in  bac- 
teria, but  that  their  numbers  decrease  as  the  soil  is  pene- 
trated, until  below  a  depth  of  one  meter  there  are  very  few. 
Remembering  that  micro-organisms  live  chiefly  upon  organic 
matter,  this  is  readily  understandable,  as  most  of  the  organic 
matter  is  upon  the  surface  of  the  soil.  Where,  as  in  the  case 
of  porous  soil  or  the  presence  of  cesspools  and  dung-heaps,  the 
decomposing  materials  are  allowed  to  penetrate  to  a  consider- 
able depth,  micro-organisms  may  occur  much  farther  below 
the  surface;  yet  they  are  rarely  found  at  any  great  depth, 
because  the  majority  of  them  require  free  oxygen  for  suc- 
cessful existence. 

The  water  of  stagnant  pools  always  teems  with  micro- 
organisms; that  of  deep  wells  rarely  contains  many  unless  it 
is  polluted  from  the  surface  of  the  earth. 

It  has  been  suggested  by  Soyka  that  currents  of  air 
passing  over  the  surface  of  liquids  might  take  up  organisms, 
but,  although  he  seemed  to  show  it  experimentally,  it  is 
not  generally  believed.  Where  bacteria  are  growing  in 
colonies  they  seem  to  remain  undisturbed  by  currents  of 

*  "Floating  Matter  in  the  Air." 

58 


Conditions  Prejudicial  to  Growth  of  Bacteria     59 

air  unless  the  surface  of  the  colony  becomes  roughened  or 
broken. 

Most  of  the  organisms  carried  about  by  the  air  are  what 
are  called  saprophytes,  and  are  harmless. 

Oxygen. — As  all  micro-organisms  must  have  oxygen 
in  order  to  live,  the  greater  number  of  them  grow  best 
when  freely  exposed  to  the  air.  Some  will  not  grow  at  all 
where  uncombined  oxygen  is  present,  but  secure  all  they 
need  by  severing  it  from  its  chemic  combinations.  These 
peculiarities  divide  bacteria  into  the 

Aerobes,  which  grow  in  the  presence  of  uncombined 
oxygen,  and  the 

Anaerobes,  which  do  not  grow  in  the  presence  of  uncom- 
bined oxygen. 

As,  however,  some  of  the  aerobic  forms  grow  almost  as 
well  without  oxygen  as  with  it,  they  are  known  as  optional 
(facultative)  anaerobes. 

As  examples  of  strictly  aerobic  bacteria  Bacillus  subtilis, 
Bacillus  aerophilus,  Bacillus  tuberculosis,  and  Bacillus 
diphtherias  may  be  given.  These  will  not  grow  if  oxygen 
is  denied  them.  The  cocci  of  suppuration,  the  bacillus  of 
typhoid  fever,  and  the  spirillum  of  cholera  grow  almost 
equally  well  with  or  without  oxygen,  and  hence  belong  to 
the  optional  anaerobes.  The  bacilli  of  tetanus  and  of 
malignant  edema,  and  the  non-pathogenic  Bacillus  butyricus, 
Bacillus  muscoides,  and  Bacillus  polypiformis,  will  not 
develop  at  all  where  any  free  oxygen  is  present,  and  hence 
are  strictly  anaerobic. 

The  higher  bacteria,  oidia,  molds  and  protozoa,  are  for  the 
most  part  aerobic  and  optional  anaerobes.  Treponema 
pallidum  seems  to  be  a  strictly  anaerobic  protozoan. 

Food. — The  bacteria  grow  best  where  diffusible  albu- 
mins are  present,  the  ammonium  salts  being  less  fitted 
to  support  them  than  their  organic  compounds.  Pros- 
kauer  and  Beck*  have  succeeded  in  growing  the  tubercle 
bacillus  in  a  mixture  containing  ammonium  carbonate 
0.35  per  cent.,  potassium  phosphate  0.15  per  cent., 
magnesium  sulphate  0.25  per  cent.,  and  glycerin  1.5 
per  cent.  Some  of  the  water  microbes  can  live  in  dis- 
tilled water  to  which  the  smallest  amount  of  organic 
matter  has  been  added;  others  require  so  concentrated 
a  medium  that  only  blood-serum  can  be  used  for  their 

*  "Zeitschrift  fiir  Hygiene,"  etc.,  Aug.  10,  1894,  vol.  xvm,  No.  i. 


60  Biology  of  Micro-organisms 

cultivation.  The  statement  that  certain  forms  of  bac- 
teria can  flourish  in  clean  distilled  water  seems  to  be 
untrue,  as  in  this  medium  the  organisms  soon  die  and 
disintegrate.  If,  however,  in  making  the  transfer,  a  drop 
of  culture  material  is  carried  into  the  water  with  the  bac- 
teria, the  distilled  water  ceases  to  be  such,  and  becomes  a 
dilute  bouillon  fitted  to  support  bacterial  life  for  a  time. 
Sometimes  a  species  with  a  preference  for  a  particular 
culture  medium  can  gradually  be  accustomed  to  another, 
though  immediate  transplantation  causes  the  death  of  the 
organism.  Sometimes  the  addition  of  such  substances 
as  glucose  and  glycerin  has  a  peculiarly  favorable  influ- 
ence, the  latter,  for  example,  enabling  the  tubercle  bacillus 
to  grow  upon  agar-agar. 

The  yeasts  grow  best  upon  media  containing  sugars,  but 
can  also  be  cultivated  upon  media  containing  diffusible 
protein  and  non-fermentable  carbohydrates  and  glycerin. 

The  molds  flourish  upon  almost  all  kinds  of  organic  matter, 
but  perhaps  attain  their  most  rapid  development  upon  media 
containing  fermentable  carbohydrates. 

The  saprophytic  and  parasitic  protozoa  live  by  osmosis  and 
absorb  through  the  ectosarc  such  substances  as  are  capable 
of  assimilation  and  nutrition.  These  forms  are  cultivable 
only  upon  media  containing  the  same  or  approximately 
the  same  proteins  as  those  to  which  they  have  been  accus- 
tomed. Thus,  to  cultivate  trypanosoma,  blood-serum 
must  be  added  to  the  media. 

The  larger  protozoa  live  upon  smaller  animal  and  vegetable 
organisms,  which  they  ingest  entire.  Such  can  only  be  arti- 
ficially cultivated  provided  the  attempt  be  made  under  con- 
ditions of  symbiosis  with  some  other  and  smaller  organism 
that  may  constitute  the  food.  Amoeba  coli  can  thus  be  cul- 
tivated in  symbiosis  with  Bacillus  typhosus. 

Moisture. — A  certain  amount  of  water  is  indispensable 
to  the  growth  of  bacteria.  The  amount  can  be  exceedingly 
small,  however,  Bacillus  prodigiosus  being  able  to  develop 
successfully  upon  crackers  and  dried  bread.  Artificial 
culture-media  should  not  be  too  concentrated;  at  least 
80  per  cent,  of  water  should  be  present. 

The  molds  and  oidia  grow  well  upon  bread  that  contains 
very  little  moisture.  Protozoa  usually  require  fluid  media. 
Pond-water  protozoa  can  only  grow  in  water,  not  in  con- 
centrated culture-media. 


Conditions  Prejudicial  to  Growth  of  Bacteria     61 

Reaction. — Should  the  pabulum  supplied  contain  an 
excess  of  either  alkali  or  acid,  the  growth  of  the  micro- 
organisms is  inhibited.  Most  true  bacteria  grow  best  in  a 
neutral  or  feebly  alkaline  medium.  There  are  exceptions 
to  this  rule,  however,  for  Bacillus  butyricus  and  Sarcina 
ventriculi  can  grow  well  in  strong  acids,  and  Micrococcus 
urea  can  tolerate  excessive  alkalinity.  Acid  media  are 
excellent  for  the  cultivation  of  molds.  Neutral  or  feebly 
alkaline  media  serve  best  for  the  cultivable  protozoa. 

Light. — Most  organisms  are  not  influenced  by  the  presence 
or  absence  of  ordinary  diffused  daylight.  The  direct 
rays  of  the  sun,  and  to  a  less  degree  the  rays  of  the  electric 
arc-light,  retard  and  in  numerous  instances  kill  bacteria.  In 
a  recent  careful  study  of  this  subject  Weinzirl*  found  that 
when  bacteria  were  placed  upon  glass  or  paper,  and  exposed 
to  the  direct  rays  of  the  sun,  without  any  covering,  most 
spore-bearing  bacteria,  including  Bacillus  tuberculosis,  B, 
diphtheriae,  B.  typhosus,  s.  cholerae  asiaticae,  B.  coli,  B.  pro- 
digiosus,  and  others  are  killed  in  from  two  to  ten  minutes. 
Certain  colors  are  distinctly  inhibitory  to  the  growth,  blue 
being  especially  prejudicial, 

Treskinskajaf  found  that  sunlight  had  a  marked  destruc- 
tive effect  upon  the  tubercle  bacillus,  and  varied  according 
to  altitude.  By  direct  sunlight  at  the  sea-level  they  were 
destroyed  in  five  hours:  at  an  altitude  of  1560  meters,  in 
three  hours.  In  winter  the  time  of  destruction  was  about 
two  hours  longer  than  in  summer.  In  diffused  daylight  the 
time  required  for  destruction  was  about  twice  as  long  as  in 
direct  sunlight.  His  experiments  were  performed  with 
pure  cultures  dried  in  a  thin  layer  upon  glass. 

Certain  chromogenic  bacteria  produce  colors  only  when 
exposed  to  the  ordinary  light  of  the  room.  Bacillus  mycoides 
roseus  produces  its  red  pigment  only  in  the  dark.  The 
virulence  of  many  pathogenic  bacteria  is  gradually  attenuated 
if  they  are  kept  in  the  light. 

Molds  and  yeasts  grow  best  in  the  dark,  so  that  in  general 
it  can  be  said  that  the  vegetable  micro-organisms,  belonging 
to  the  fungi  and  having  no  chlorophyl,  need  no  light  and  are 
injured  rather  than  benefited  by  it. 

The  pathogenic  protozoa  have  not  been  particularly  studied 

*  "  Centralbl.  f.  Bakt.  u.  Parasitenk.  Ref.,"  xi/ra,  Nos.  22-24,  P-  68l« 
f  "  Jour.    Infectious    Diseases,"  vol.  iv,  1907,   Supplement,  No.  3, 
p.  128. 


62  Biology  of  Micro-organisms 

with  reference  to  light.  Non-pathogenic  water  protozoa 
love  the  light  and  die  in  the  dark. 

Electricity,  X-rays,  etc. — Powerful  currents  of  electricity 
passed  through  cultures  have  been  found  to  kill  the  organ- 
isms and  change  the  reaction  of  the  culture-medium;  rapidly 
reversed  currents  of  high  intensity,  to  destroy  the  patho- 
genesis  of  the  bacteria  and  transform  their  toxic  products 
into  neutralizing  bodies  (antitoxin?).  Attention  has  been 
called  to  this  subject  by  Smirnow,  d'Arsonval  and  Charin, 
Bolton  and  Pease,  Bonome  and  Viola,  and  others. 

An  interesting  contribution  upon  the  "  Effect  of  Direct, 
Alternating,  Tesla  Currents  and  X-rays  on  Bacteria  "  was 
made  by  Zeit,*  whose  conclusions  are  as  follows: 

1.  A  continuous  current  of  260  to  320  milliamperes  passed  through 
bouillon  cultures  kills  bacteria  of  low  thermal  death-points  in  ten  min- 
utes by  the  production  of  heat  (98.5°  C.).     The  antiseptics  produced 
by  electrolysis  during  this  time  are  not  sufficient  to  prevent  the  growth 
of  even  non-spore-bearing  bacteria.     The  effect  is  a  purely  physical  one. 

2.  A  continuous  current  of  48  milliamperes  passed  through  bouillon 
cultures  for  from  two  to  three  hours  does  not  kill  even  non-resistant 
forms  of  bacteria.     The  temperature  produced  by  such  a  current  does 
not  rise  above  37°  C.,  and  the  electrolytic  products  are  antiseptic,  but 
not  germicidal. 

3.  A  continuous  current  of  100  milliamperes  passed  through  bouillon 
cultures  for  seventy-five  minutes  kills  all  non-resistant  forms  of  bacteria 
even  if  the  temperature  is  artificially  kept  below  37°  C.      The  effect  is 
due  to  the  formation  of  germicidal  electrolytic  products  in  the  culture. 
Anthrax  spores  are  killed  in  two  hours.     Subtilis  spores  were  still  alive 
after  the  current  was  passed  for  three  hours. 

4.  A  continuous  current   passed   through   bouillon  cultures  of  bac- 
teria produces  a  strongly  acid  reaction  at  the  positive  pole,  due  to  the 
liberation  of  chlorin  which  combines  with  oxygen  to  form  hypochlorous 
acid.     The  strongly  alkaline  reaction  of  the  bouillon  culture  at   the 
negative  pole  is  due  to  the  formation  of  sodium  hydroxid  and  the  libera- 
tion of  hydrogen  in  gas  bubbles.     With  a  current  of  100  milliamperes 
for  two  hours  it  required  8.82  milligrams  of  H2SO4  to  neutralize  i  c.c.  of 
the  culture  fluid  at  the  negative  pole,  and  all  the  most  resistant  forms 
of  bacteria  were  destroyed  at  the  positive  pole,  including  anthrax  and 
subtilis  spores.     At  the  negative  pole  anthrax  spores  were  killed  also, 
but  subtilis  spores  remained  alive  for  four  hours. 

5.  The  continuous  current  alone,  by  means  of  Du  Bois-Reymond's 
method  of  non-polarizing  electrodes,  and  exclusion  of  chemic  effects  by 
ions   in   Kruger's   sense,  is  neither   bactericidal    nor   antiseptic.      The 
apparent  antiseptic  effect  on  suspension  of  bacteria  is  due  to  electric 
osmosis.     The  continuous  electric  current  has  no  bactericidal  nor  anti- 
septic properties,  but  can  destroy  bacteria  only  by  its  physical  effects 
(heat)  or  chemic  effects  (the  production  of  bactericidal  substances  by 
electrolysis). 

6.  A  magnetic  field,  either   within  a  helix  of    wire  or  between   the 
poles  of   a  powerful  electromagnet,  has  no  antiseptic  or   bactericidal 
effects  whatever. 

*  "Jour   Amer.  Med.  Assoc.,"  Nov.  30,  1901. 


Conditions  Prejudicial  to  Growth  of  Bacteria     63 

7.  Alternating  currents  of  a  3-inch  Ruhmkorff  coil  passed  through 
bouillon  cultures  for  ten  hours  favor  growth  and  pigment  production. 

8.  High-frequency,    high  potential  currents — Tesla   currents — have 
neither  antiseptic  nor  bactericidal  properties  when  passed  around  a  bac- 
terial suspension  within  a  solenoid.      When  exposed  to  the  brush  dis- 
charges, ozone  is  produced  and  kills'  the  bacteria. 

9.  Bouillon  and  hydrocele-fluid  cultures  in  test-tubes  of  non-resistant 
forms  of  bacteria  could  not  be  killed  by  Rontgen  rays  after  forty-eight 
hours'  exposure  at  a  distance  of  20  mm.  from  the  tube. 

10.  Suspensions  of  bacteria  in  agar  plates  and  exposed  for  four  hours 
to  the  rays,  according  to  Rieder's  plan,  were  not  killed. 

1 1 .  Tubercular  sputum  exposed  to  the  Rontgen  rays  for  six  hours, 
at  a  distance  of  20  mm.  from  the  tube,  caused  acute  miliary  tubercu- 
losis of  all  the  guinea-pigs  inoculated  with  it. 

12.  Rontgen  rays  have  no  direct  bactericidal  properties.      The  clin- 
ical results  must  be  explained  by  other  factors,  possibly  the  production 
of  ozone,  hypochlorous  acid,  extensive  necrosis  of  the  deeper  layers  of 
the  skin,  and   phagocytosis.     The  action  of  the  x-rays  upon  bacteria 
has  been  investigated  by  Bonome  and  Gros,*  Pott,f  and  others.     When 
the  cultures  are  exposed  to   their  action  for  prolonged  periods,  their 
vitality  and  virulence  seem  to  be  slightly  diminished.      They  are  not 
killed  by  the  x-rays. 

Movement. — Rest  seems  to  be  the  condition  best  adapted 
for  micro-organismal  development.  Slow-flowing  movements 
do  not  have  much  inhibitory  action,  but  violent  agitation, 
as  by  shaking  a  culture  in  a  machine,  may  hinder  or  prevent 
it.  This  explains  why  rapidly  flowing  streams,  whose  cur- 
rents are  interrupted  by  falls  and  rapids,  should,  other 
things  being  equal,  furnish  a  better  drinking-water  than  a 
deep,  still-flowing  river. 

Galli- Valerio  t  has  shown,  however,  that  agitation  does 
not  inhibit  the  growth  of  the  anthrax,  typhoid  or  colon 
bacilli  or  the  pneumococcus,  but  sometimes  facilitates  it. 

Association. — Symbiosis  is  the  vital  association  of  different 
species  of  micro-organisms  by  which  mutual  benefit  to  one 
or  the  other  is  brought  about.  Antibiosis  is  an  association 
detrimental  to  one  of  the  associated  organisms.  Bacterial 
growth  is  greatly  modified  by  the  association  of  different 
species.  Coley  found  the  streptococcus  more  active  when 
combined  with  Bacillus  prodigiosus;  Pawlowski,  that  mixed 
cultures  of  Bacillus  anthracis  and  Bacillus  prodigiosus  were 
less  virulent  than  pure  cultures  of  anthrax;  Meunier,§  that 

*  "Giornal.  med.  del  Regis  Esercito,"  an  45,  u.  6. 

f  "Lancet,"  vol.  n,  No.  21,  1897. 

t  "  Centralbl.  f.  Bakt.,"  etc.,  I  Orig.  4,  xxxvn,  Sept.  23,  1904, 
p.  151. 

§  Societe  de  Biologic,  Seance  du  n  Juin,  1898,  "La  Semaine  medi- 
cale,"  June  15,  1898. 


64  Biology  of  Micro-organisms 

when  the  influenza  bacillus  of  Pfeiffer  is  inoculated  upon 
blood  agar  together  with  Staphylococcus  aureus  its  growth 
is  favored  by  a  change  which  the  staphylococci  bring  about 
in  the  hemoglobin. 

A  similar  advantageous  association  has  been  pointed  out 
by  Sanarelli,  who  found  that  Bacillus  icteroides  grows  best 
and  retains  its  vitality  longest  when  grown  in  company  with 
certain  of  the  molds. 

Rarely,  the  presence  of  one  species  of  micro-organism 
entirely  eradicates  another.  Hankin*  found  that  Micro- 
coccus  ghadialli  destroyed  the  typhoid  and  colon  bacilli, 
and  suggested  the  use  of  this  coccus  to  purify  waters  pol- 
luted with  typhoid. 

An  interesting  experimental  study  of  the  bacterial  an- 
tagonisms with  special  reference  to  the  Bacillus  typhosus, 
that  the  student  should  read,  is  by  W.  D.  Frost,  and  appears 
in  the  "  Journal  of  Infectious  Diseases,"  1904,  i,  p.  599. 

Temperature. — According  to  Frankel,  bacteria  will  rarely 
grow  below  16°  and  above  40°  C.,  but  Fliigge  has  shown  that 
Bacillus  subtilis  will  grow  very  slowly  at  6°  C.;  at  12.5°  C. 
fission  does  not  take  place  oftener  than  every  four  or  five 
hours;  at  25°  C.  fission  occurs  every  three-quarters  of  an  hour, 
and  at  30°  C.  about  every  half-hour. 

The  temperature  at  which  micro-organisms  grow  best  is 
known  as  the  optimum,  the  lowest  temperature  at  which 
they  continue  active  as  the  minimum,  the  highest  that  can 
be  endured  the  maximum. 

A  few  forms  of  bacteria  grow  at  very  high  temperatures 
(6o°-7o°  C.),  and  are  described  as  thermophilic .  They  are 
found  in  manure  piles  and  in  hot  springs.  Tsiklinskyf  has 
described  two  varieties  of  actinomyces  and  a  mold  that  he 
cultivated  from  earth  and  found  able  to  grow  well  at  48°  to 
68°  C.,  though  not  at  all  at  the  temperature  of  the  room. 

Most  bacteria  are  killed  by  temperatures  above  60°  to 
75°  C.,  but  their  spores  can  resist  boiling  water  for  some 
minutes,  though  killed  by  dry  heat  if  exposed  to  150°  C.  for 
an  hour  or  to  175°  C.  for  from  five  to  ten  minutes. 

The  resistance  of  low  forms  of  life  to  low  temperatures  is 
most  astonishing.  Some  adult  bacteria  and  most  spores 
seem  capable  of  resisting  almost  any  degree  of  cold.  Ravenel  % 

*  "Brit.  Med.  Jour.,"  Aug.  14,  1897,  p.  418. 

t  "Russ.  Archiv  f.  Path.,"  etc.,  Bd.  v,  June,  1898. 

t  "The  Medical  News,"  June  10,  1899. 


Conditions  Prejudicial  to  Growth  of  Bacteria     65 

exposed  anthrax  spores  to  the  action  of  liquid  air  for  three 
hours;  diphtheria  bacilli,  for  thirty  minutes ;  typhoid  bacilli, 
for  sixty  minutes ;  and  Bacillus  prodigiosus,  for  sixty  minutes, 
the\emperature  of  the  cultures  being  reduced  to  about  — 140° 
C.,  yet  in  no  case  was  the  vegetative  capability  of  all  of  the 
bacteria  destroyed,  and  when  transferred  to  fresh  culture 
bouillon  they  grew  normally.  His  researches  corroborate 
those  of  Pictet  and  Yung  and  others. 

To  say  that  bacteria  are  not  injured  by  cold  is  a  mistake, 
as  Sedgwick  and  Winslow*  have  found  that  when  typhoid 
bacilli  are  frozen,  the  greater  number  of  them  are  destroyed, 
and  that  subsequent  development  of  the  frozen  cultures 
takes  place  from  the  few  surviving  organisms. 

Bacteria  usually  grow  best  at  the  temperature  of  a  com- 
fortably heated  room  (17°  C.),  and  are  not  affected  by  its 
occasional  slight  variations.  Some,  chiefly  the  pathogenic 
forms,  are  not  cultivable  except  at  the  temperature  of  the 
body  (37°  C.);  others,  like  the  tubercle  bacillus,  grow  best 
at  a  temperature  a  little  above  that  of  the  normal  body. 

The  temperature  endurance  of  the  molds  resembles  that 
of  the  bacteria.  The  mycelia  are  killed  at  temperatures  of 
60°  C.  and  over,  but  their  spores  endure  100°  C.  The 
yeasts  and  oi'dia,  that  have  no  resisting  spores,  are  killed  at 
about  60°  C.  The  protozoa  are  still  more  sensitive  to  heat 
variations  than  the  plant  organisms  and  are  killed  by  less 
extreme  variations.  Here  again,  however,  the  encysted  pro- 
tozoa endure  greater  variations  than  the  active  organisms. 

Effect  of  Chemic  Agents. — The  presence  of  chemic  agents, 
especially  certain  of  the  mineral  salts,  in  an  otherwise  per- 
fectly suitable  medium  may  completely  inhibit  the  develop- 
ment of  bacteria,  and  if  added  to  grown  cultures  in  greater 
concentration,  destroy  them.  Such  substances  are  spoken 
of  as  antiseptics  in  the  former,  germicides  in  the  latter  case. 
Bichlorid  of  mercury  and  carbolic  acid  are  the  most  familiar 
examples  of  germicides. 

Though  these  agents  are  supposed  to  operate  in  definite 
concentrations  with  almost  unvarying  result,  Trambustif 
found  it  possible  to  produce  a  tolerance  to  a  certain  amount 
of  bichlorid  of  mercury  by  cultivating  Friedlander's  bacillus 
upon  culture-media  containing  gradually  increasing  amounts 

:"Centralbl.   f.    Bakt.   u.    Parasitenk.,"   etc.,   May   26,    190x3,   Bd. 
xxvu,  Nos.  1 8,  19,  p.  684. 

f"Lo  Sperimentale,"   1893-94. 
5 


66  Biology  of  Micro-organisms 

of  the  salt,  until  from  1-15,000,  which  inhibit  ordinary  cul- 
tures, it  could  accommodate  itself  to  1-2000. 

The  various  chemic  agents  act  in  different  ways  upon  the 
micro-organisms.  Thus,  they  may  combine  with  the  pr'oto- 
plasm  to  make  a  new  and  no  longer  vital  cornpound ;  or,  they 
may  coagulate  or  dissolve  or  dehydrate  or  oxidize  the  proto- 
plasm to  a  destructive  extent. 

The  addition  of  chemic  agents  to  solutions  containing 
micro-organisms  also  changes  the  osmotic  pressure.  When 
an  active  organism  is  living  in  its  normal  environment,  it 
contains  within  its  plasm  a  greater  concentration  of  solutes 
than  are  to  be  found  in  the  surrounding  fluid.  Under  these 
circumstances  the  pressure  on  the  inside  of  the  ectosarc 
or  other  cell  membrane  is  greater  than  that  on  the  outer 
side,  and  the  cell  is  in  a  state  of  turgor.  If  now  salts  are  added 
so  that  the  solutes  on  the  outside  exceed  those  on  the  inside, 
water  is  drawn  out  and  the  protoplasm  is  made  to  shrink  or 
condense.  According  to  the  degree  of  this  change  the 
organism  will  be  embarrassed,  made  impotent,  or  destroyed. 
On  the  other  hand,  when  micro-organisms  have  enjoyed 
a  concentrated  medium  like  blood-serum  and  are  suddenly 
transferred  to  distilled  water,  so  much  water  may  be  sud- 
denly drawn  into  their  protoplasm  that  they  swell  up  and 
may  burst  and  go  to  pieces.  This  is  particularly  true  of  the 
delicate  protozoa  like  the  trypanosoma. 

Metabolism. — According  to  their  activities,  micro- 
organisms are  classed  as — 

Zymogens,  when  they  cause  fermentation. 

Saprogens,  when  they  cause  putrefaction. 

Chromogens,  when  they  produce  colors. 

Photogens,  when  they  phosphoresce. 

Aerogens,  when  they  evolve  gas. 

Pathogens,  when  they  cause  disease. 

The  metabolic  activities  of  micro-organisms  occasion  many 
well-known  changes  in  nature.  Thus,  it  is  through  their 
energies  that  by  fermentative  and  putrefactive  changes 
organic  matter  is  gradually  transformed  from  complex  to 
simple  compounds.  It  is  by  the  energy  of  bacteria  that  foul 
waters  are  gradually  purified,  and  while  it  is  true  that  the 
presence  of  large  numbers  of  bacteria  in  water  detracts  from 
its  potability,  the  very  bacteria  that  cause  its  condemnation 
ultimately  effect  its  purification  by  exhausting  the  organic 
matter  it  contains  in  their  own  nutrition.  In  the  modern 


Fermentation  67 

treatment  of  sewage  by  the  "septic  tank  "  method,  the 
organic  matter  contained  in  the  water  is  consumed  through 
the  agency  of  anaerobic  and  aerobic  bacteria,  until  its  con- 
sumption leaves  the  water  once  more  clear  and  pure,  the  no 
longer  useful  bacteria  dying  out  as  the  nutrition  becomes  ex- 
hausted. 

The  promptness  with  which  bacteria  attack  organic 
matter  is  seen  in  the  changes  brought  about  in  foods,  some 
of  which  are  ruined  in  flavor  or  quality,  though  others 
are  thought  to  be  improved.  Thus,  the  flavor  of  butter, 
sausage,  and  cheese,  the  aroma  of  wines,  and  many  other 
important  gustatory  characteristics  of  our  foods  depend 
solely  upon  the  activity  of  bacteria  or  other  micro-organisms. 

Many  of  these  activities  are  harmless,  and,  indeed,  ad- 
vantageous, though  the  fact  that  they  are  not  infrequently 
accompanied  by  chemic  changes,  some  of  which  are  poison- 
ous, makes  it  necessary  to  watch  and  time  their  operations 
lest  acridity,  acidity,  insipidity,  or  toxicity  of  the  food  re- 
place the  desired  effect. 

Briefly  considered,  the  best-known  phenomena  resulting 
from  micro-organismal  energy  are  as  follows: 

Fermentation. — Fermentation  is  a  chemic  transforma- 
tion of  carbohydrates  resulting  from  the  activity  of  micro- 
organismal  enzymes.  The  alcoholic  fermentation,  which  is 
a  familiar  phenomenon  to  the  layman  as  well  as  to  the  brewer 
and  chemist,  depends  upon  the  activity  of  an  yeast-plant,  one 
of  the  saccharomyces  fungi  by  which  the  sugar  is  broken  up 
into  alcohol  and  carbon  dioxid,  with  some  glycerin  and  other 
by-products.  The  following  equation  shows  the  chief  changes 
produced : 

C6H1206  2C2H5OH     -f     2C02 

Sugar.  Alcohol.  Carbon  dioxid. 

There  are  also  several  bacteria  which  produce  the  acetic 
fermentation,  though  it  is  generally  attributed  to  Bacillus 
aceticus.  There  are  two  equations  to  express  this  fermen- 
tation : 

I.  CH2CH2OH     +     O      =     CH3CHO     +     H2O 
Alcohol.  Oxygen.  Aldehyd.  Water. 

II.  CH,CHO     +     O      =     CH3COOH 

Aldehyd.  Oxygen.  Acetic  acid. 

A  number  of  different  bacilli  seem  capable  of  converting 
milk-sugar  into  lactic  acid,  though  Bacillus  acidi  lactici  is 


68  Biology  of  Micro-organisms 

the  best  known  and  most  active  acid  producer.  The  butyric 
fermentation  generally  due  to  Bacillus  butyricus  may  also 
be  caused  by  other  bacilli.  (For  an  exact  description  of  the 
chemistry  of  the  fermentations  reference  must  be  made  to 
special  text-books.*)  The  lactic  acid  and  butyric  acid  fer- 
mentation, have  the  following  equations: 

I.  C»HMQu     +     H2Q     =     C0H1202     +     C,Hi,Ofl 
Lactose  or  milk  sugar.  Galactose.  Dextrose. 

II.  CoHiA     =     2CzU,O3 
Galactose.  Lactic  acid. 

III.  C6H,206     =     C4H80^    +     C02     +     2H2 
Galactose.  Butyric  acid. 

Putrefaction. — Putrefaction  is  a  chemic  disintegration 
of  nitrogenous  compounds  resulting  from  the  activity  of 
micro-organisms.  The  first  step  in  the  process  seems  to 
be  the  transformation  of  the  albumins  into  peptones,  then 
the  splitting  up  of  the  peptones  into  gases,  acids,  bases, 
and  salts.  Both  fermentative  and  putrefactive  processes 
apparently  take  place  through  the  agency  of  enzymes. 
In  the  process  the  innocuous  albumins  are  frequently  changed 
to  toxalbumins,  and  sometimes  to  peculiar  putrefactive  al- 
kaloids known  as  ptomains. 

Ptomains. — Vaughan  and  Novy  define  a  ptomain  as  "  a 
chemical  compound,  basic  in  character,  formed  by  the  action 
of  bacteria  on  organic  matter."  The  chemistry  of  these 
bodies  is  very  complex,  and  for  a  satisfactory  description 
of  them  Vaughan  and  Novy's  bookf  is  excellent. 

Ptomains  probably  play  but  a  small  part  in  pathologic 
conditions.  They  are  formed  almost  exclusively  outside  of 
the  living  body,  and  only  become  a  source  of  danger  when 
ingested  with  the  food.  It  is  supposed  that  cases  of  ice- 
cream and  cheese-poisoning  are  usually  due  to  tyrotoxicon, 
a  ptomain  produced  by  the  putrefaction  of  the  protein  sub- 
stances of  the  milk  before  it  is  frozen  into  ice-cream  or 
made  into  cheese.  The  safeguard  is  to  freeze  the  milk  only 

*See  "Enzymes  and  Their  Applications,"  by  Jean  Effront,  trans- 
lated by  S.  C.  Prescott,  New  York,  1902;  "Micro-organisms  and  Fer- 
mentation," by  Alfred  Jorgensen,  translated  by  A.  K.  Miller  and 
A.  E.  Lennholm,  London,  1900;  and  the  many  writings  of  Christian 
Hansen. 

f  "Ptomaines  and  Leucomaines,"  1888;  "Cellular  Toxins,"  1902. 


Production  of  Gases 


when  perfectly  fresh  and  avoid  mixing  the  milk,  cream, 
sugar,  and  flavoring  substances,  and  allowing  the  mixture 
to  stand  for  some  time  beforehand. 

The  occasional  cases  of  "  Fleischver-giftung,"  "meat- 
poisoning,"  or  "  Botulismus,"  are  due  to  the  development  of 
toxic  ptomains  in  consequence  of  the 
growth  of  certain  bacteria  (Bacillus 
botulinus)  in  the  meat.  Kaensche* 
carefully  investigated  the  subject,  and 
gives  a  synoptic  table  containing  all  the 
described  bacteria  of  this  class.  His 
researches  show  that  there  are  at  least 
three  different  bacilli  whose  growth 
causes  the  development  of  poisonous 
ptomains  in  meat.  In  general  these  or- 
ganisms resemble  Bacillus  coli. 

With  the  increase  of  knowledge  upon 
the  toxic  character  of  the  bacteria  them- 
selves, the  importance  of  the  toxic  pto- 
mains has  diminished,  until  at  present 
we  have  come  to  regard  them  as  very 
rare  causes  of  disease. 

Production  of  Gases. — Various  gases 
are  given  off  during  decomposition  and 
fermentation,  among  them  being  CO2, 
H2S,  NH4,  H,  CH4,  and  others.  Gases 
produced  by  aerobic  bacteria  usually  fly  off  from  the  sur- 
face of  the  culture  unnoticed,  but  if  the  bacterium  be 
anaerobic  and  develop  at  the  lower  part  of  a  tube  of 
culture  media,  a  visible  bubble  of  gas  is  usually  formed 
about  the  colonies.  Such  gas  bubbles  are  almost  invariably 
present  in  cultures  of  the  bacilli  of  tetanus  and  malignant 
edema. 

To  quantitatively  determine  the  gas-production,  some 
form  of  the  Smith  fermentation-tube  is  most  convenient. 
The  tube  is  filled  with  bouillon  containing  some  sugar, 
sterilized  as  usual,  inoculated,  and  stood  aside  to  grow. 
As  the  gases  form,  the  bubbles  ascend  and  accumulate  in 
the  closed  arm.  In  estimating  quantitatively,  one  must 
be  careful  that  the  tube  is  not  so  constructed  as  to  allow 
the  gas  to  escape  as  well  as  to  ascend  into  the  main 
reservoir. 

*  "Zeitschrift  fiir  Hygiene,"  etc.,  Bd.  xxn,  Heft  i,  June  25,  1896. 


Fig.  1 6. — Smith's  fer- 
mentation-tube. 


70  Biology  of  Micro-organisms 

For  the  determination  of  the  nature  of  the  gases  produced, 
Theobald  Smith  has  recommended  the  following  method : 

"The  bulb  is  completely  filled  with  a  2  per  cent,  solution 
of  sodium  hydroxid  (NaOH)  and  tightly  closed  with  the 
thumb.  The  fluid  is  shaken  thoroughly  with  the  gas  and 
allowed  to  flow  back  and  forth  from  the  bulb  to  the  closed 
branch,  and  the  reverse  several  times  to  insure  intimate 
contact  of  the  CO2  with  the  alkali.  Lastly,  before  removing 
the  thumb  all  the  gas  is  allowed  to  collect  in  the  closed 
branch  so  that  none  may  escape  when  the  thumb  is  removed. 
If  CO2  be  present,  a  partial  vacuum  in  the  closed  branch 
causes  the  fluid  to  rise  suddenly  when  the  thumb  is  removed. 
After  allowing  the  layer  of  foam  to  subside  somewhat  the 
space  occupied  by  gas  is  again  measured,  and  the  difference 
between  this  amount  and  that  measured  before  shaking 
with  the  sodium  hydroxid  solution  gives  the  proportion  of 
CO2  absorbed.  The  explosive  character  of  the  residue  is 
determined  as  follows:  The  cotton  plug  is  replaced  and 
the  gas  from  the  closed  branch  is  allowed  to  flow  into  the 
bulb  and  mix  with  the  air  there  present.  The  plug  is  then 
removed  and  a  lighted  match  inserted  into  the  mouth  of 
the  bulb.  The  intensity  of  the  explosion  varies  with  the 
amount  of  air  present  in  the  bulb.  The  relative  propor- 
tion of  gases  resulting  from  the  fermentation  is  frequently 
of  importance  for  the  differential  diagnosis  of  related  bac- 

TT 

teria.     Smith  has  designated  this  relation  of  ^o-  as  the  'gas 
formula.'      The   colon   bacillus  has   a   gas   formula   corre- 

TT 

spending  to   ^  —  f .     Other   aerogenic   bacilli   sometimes 
show  a  formula  ^j  =  ^." 

Liquefaction  of  Gelatin. — As  certain  organisms  grow  in 
gelatin,  the  medium  becomes  partly  or  entirely  liquefied. 
This  peculiarity  is  apparently  independent  of  any  other 
property  of  the  organisms,  and  is  manifested  alike  by  patho- 
genic and  non-pathogenic  forms.  The  liquefaction  is  sup- 
posed to  be  dependent  upon  a  form  of  peptonization.  Bit- 
ter* and  Sternbergf  have  shown  that  if  from  a  culture  in 
which  liquefaction  has  taken  place  the  bacteria  be  removed 
by  filtration,  the  filtrate  will  retain  the  power  of  liquefying 
gelatin,  showing  the  property  is  not  resident  in  the  bacteria, 
but  in  some  substance  in  solution  in  their  excreted  products. 

*  "  Archiv  Fur  Hygiene,"  1886,  Heft  2. 
t"  Medical  News,"  1887,  No.  14. 


Chromogenesis  71 

These  products  were  described  as  "  tryptic  enzymes  "  by 
Fermi,*  who  found  that  heat  destroyed  them.  Mineral 
acids  seem  to  check  their  power  to  act  upon  gelatin.  For- 
malin renders  the  gelatin  insoluble.  Some  of  the  bacteria 
liquefy  the  gelatin  in  such  a  peculiar  and  characteristic 
manner  as  to  make  the  appearance  a  valuable  guide  for  the 
differentiation  of  species. 

Production  of  Acids  and  Alkalies — Under  the  head 
of  "Fermentation"  the  formation  of  acetic,  lactic,  and 
butyric  acids  has  been  discussed.  Formic,  propionic,  baldri- 
anic,  palmitic,  and  margaric  acids  also  result  from  microbic 
metabolism.  As  the  acidity  progresses,  it  impedes,  and 
ultimately  completely  inhibits,  the  activity  of  the  organisms. 
The  cultivation  of  the  bacteria  in  milk  to  which  litmus  or 
lacmoid  has  been  added  is  a  convenient  method  for  detecting 
changes  of  reaction.  Rosolic  acid  solutions  may  also  be 
used,  the  acid  converting  the  red  into  an  orange  color. 
Neutral  red  is  also  much  employed  for  this  purpose,  the 
acids  turning  it  yellow. 

The  quantitative  estimation  of  changes  in  reaction  can 
be  best  made  by  titration,  and  the  fermentation-tube  culture 
can  be  employed  for  the  purpose.  The  contents  of  the  bulb 
and  branch  should  be  shaken  together,  a  measured  quan- 
tity withdrawn,  and  titration  with  ^  sodium  hydroxid,  or 
-  hydrochloric  acid,  performed. 

The  alkali  most  frequently  formed  by  bacterial  growth 
is  ammonium,  which  is  set  free  from  its  combinations, 
and  either  flies  off  as  a  gas  or  forms  new  combinations 
with  acids  simultaneously  formed.  Some  bacteria  produce 
acids  only,  some  alkalies  only,  others  both  acids  and  alka- 
lies. Both  acids  and  the  alkalies,  when  in  excess,  serve  to 
check  the  further  activity  of  the  micro-organisms. 

Chromogenesis. — Bacteria  that  produce  colored  colo- 
nies or  impart  color  to  the  medium  in  which  they  grow  are 
called  chromogenic;  those  producing  no  color,  non-chromo- 
genic.  Most  chromogenic  bacteria  are  saprophytic  and  non- 
pathogenic.  Some  of  the  pathogenic  forms,  as  Staphylo- 
coccus  pyogenes  aureus,  are,  however,  color  producers.  It 
seems  more  likely  that  certain  chromogenetic  substances 
unite  with  constituents  of  the  culture  medium  to  pro- 
duce the  colors  than  that  the  bacteria  form  the  actual 

*"Centralbl.  f.  Bakt.,"  etc.,  1891,  Bd.  x,  p.  401. 


72  Biology  of  Micro-organisms 

pigments;  but,  as  Galeotti  *  has  shown,  there  are  two  kinds 
of  pigment,  one  being  soluble,  readily  saturating  the  cul- 
ture medium,  as  the  pyocyanin  and  fluorescin  of  Bacillus 
pyocyaneus,  the  other  insoluble,  not  tingeing  the  solid  cul- 
ture media,  but  retained  in  the  colonies,  like  the  pigment 
of  Bacillus  prodigiosus.  The  pigments  are  found  in  great- 
est intensity  near  the  surface  of  a  bacterial  mass.  The 
coloring  matter  never  occupies  the  cytoplasm  of  the  bac- 
teria (except  Bacillus  prodigiosus,  in  whose  cells  occasional 
pigment-granules  may  be  seen),  but  occurs  as  an  inter- 
cellular deposit. 

Almost  all  known  colors  are  formed  by  different  bacteria. 
One  bacterium  will  sometimes  elaborate  two  or  more  colors ; 
thus,  Bacillus  pyocyaneus  produces  pyocyanin  and  fluores- 
cin, both  being  soluble  pigments — one  blue,  the  other  green. 
Gessard  f  has  shown  that  when  Bacillus  pyocyaneus  is  culti- 
vated upon  white  of  egg,  it  produces  only  the  green  fluor- 
escent pigment,  but  if  cultivated  in  pure  peptone  solution 
it  produces  only  the  blue  pyocyanin.  His  experiments  prove 
the  very  interesting  fact  that  for  the  production  of  fluor- 
escin it  is  necessary  that  the  culture  medium  contain  a  defi- 
nite amount  of  a  phosphatic  salt.  Sometimes,  when  an 
organism  produces  two  pigments,  one  is  soluble,  the 
other  insoluble,  so  that  the  colony  will  appear  one 
color,  the  medium  upon  which  it  grows  another.  I  once 
found  an  interesting  coccus,  |  with  this  peculiarity,  upon 
the  conjunctiva.  It  formed  a  brilliant  yellow  colony 
upon  the  surface  of  agar-agar,  but  colored  the  agar-agar 
itself  a  beautiful  violet.  In  this  case  the  yellow  pig- 
ment was  insoluble,  the  violet  pigment  very  soluble  and 
diffusible  through  the  jelly.  Some  organisms  will  only  pro- 
duce pigments  in  the  -light;  others,  as  Bacillus  mycoides 
roseus,  only  in  the  dark.  Some  produce  them  only  at  the 
room  temperature,  but,  though  growing  luxuriantly  in  the 
incubator,  refuse  to  produce  pigments  at  so  high  a  tem- 
perature. Thus,  Bacillus  prodigiosus  produces  a  brilliant 
red  color  when  growing  at  the  temperature  of  the  room, 
but  is  colorless  when  grown  in  the  incubator.  The  reaction 
of  the  culture  medium  is  also  of  much  importance  in  this 

*  "Lo  Sperimentale,"  1892,  XLVI,  Fasc.  m,  p.  261. 
f  "Ann.  de  1'Inst.  Pasteur,"  1892,  pp.  810-823. 
t  See  Norris  and  Oliver,  "System  of  Diseases  of  the  Eye,"  vol.  n,  p. 
489,  and  "University  Medical  Magazine,"  Sept.,  1895. 


Production  of  Aromatics  73 

connection.  Thus,  Bacillus  prodigiosus  produces  an  intense 
scarlet-red  color  upon  alkaline  and  neutral  media,  but  is 
colorless  or  pinkish  upon  slightly  acid  media.  Some  of  the 
pigments — perhaps  most  of  them — are  formed  only  in  the 
presence  of  oxygen. 

Production  of  Odors. — Gases,  such  as  H2S  and  NH4,  and 
acids,  butyric  and  acetic  acids,  have  sufficiently  character- 
istic odors.  There  are,  however,  a  considerable  number  of 
pungent  odors  which  seem  to  arise  from  independent  odor- 
iferous principles.  Many  of  them  are  extremely  un- 
pleasant, as  that  of  the  tetanus  bacillus.  The  odors  seem 
to  be  peculiar  individual  characteristics  of  the  organisms. 

Production  of  Phosphorescence. — Cultures  of  Bacillus 
phosphorescens  and  numerous  other  organisms  are  dis- 
tinctly phosphorescent.  So  much  light  is  sometimes  given 
out  by  gelatin  cultures  of  these  bacteria  as  to  enable  one  to 
see  the  face  of  a  watch  in  a  dark  room.  Gorham  found 
the  photogenesis  most  marked  when  the  organisms  are  grown 
in  alkaline  media  at  room  temperature.  Most  of  the  phos- 
phorescent bacteria  are  found  in  sea-water,  and  are  best 
cultivated  in  sea-water  gelatin.  Some  are  familiar  to 
butchers  through  the  phosphorescence  they  cause  on  the 
surface  of  fresh  meats. 

Production  of  Aromatics. — Phenol,  kresol,  hydrochinone, 
hydro  paracumaric  acid,  and  paroxypheny  lie-ace  tic  acid 
are  by  no  means  uncommon  products  of  bacteria.  The 
most  important  is  indol,  which  was  at  one  time  thought  to 
be  peculiar  to  the  cholera  spirillum,  but  is  now  known  to 
be  produced  by  many  other  bacteria.  The  best  method  of 
testing  for  it  is  that  of  Salkowski,*  known  as  the  nitroso- 
indol  reaction.  To  perform  it,  10  c.c.  of  the  fluid  to  be 
tested  receive  an  addition  of  10  drops  of  concentrated  sul- 
phuric acid.  The  mixture  is  shaken  in  a  test-tube.  A 
few  cubic  centimeters  of  a  0.02  per  cent,  solution  of  potas- 
sium nitrite  are  then  allowed  to  flow  down  the  side  of  the 
tube.  If  indol  is  present,  a  purple-red  color  develops  at 
the  junction  of  the  two  fluids,  f  McFarland  and  Small  J 
have  found  that  the  intensity  of  this  color  corresponds  to 
the  quantity  of  indol  present,  and  that  quantitative  tests 
can  be  made  by  means  of  a  comparative  color  test  series. 

t  "Zeitschrift  f.  physiol.  Chemie,"  vm,  p.  417. 

t  See  Grubs  and  Francis,  "Bull,  of  the  Hyg.  Laboratory,"  No.  7,  1902. 

t  "Trans,  of  the  American  Public  Health  Association,"  1905. 


74  Biology  of  Micro-organisms 

The  Formation  of  Nitrates. — A  process  of  fundamen- 
tal importance  is  carried  on  by  certain  lowly  bacteria  of 
the  soil.  Since  plants  are  unable  to  assimilate  the  free 
nitrogen  of  the  air,  but  must  obtain  this  element  from 
the  soil  in  the  form  of  some  soluble  compound,  and  since 
there  is  a  relatively  limited  amount  of  combined  nitrogen 
in  the  world,  it  becomes  of  the  last  importance  that  the 
supplies  which  are  continually  withdrawn  from  the  soil 
should  be  replaced  by  the  nitrogen  liberated  in  the  decay 
of  organic  material.  This  nitrogen,  after  a  series  of  putre- 
factive changes  have  occurred,  appears  as  ammonia.  The 
odor  of  this  gas  is  often  plainly  perceptible  about  manure 
heaps.  In  this  form  nitrogen  is  poorly  adapted  for  use  by 
plants,  and  moreover  may  be  easily  dissipated.  An  ex- 
tensive further  process  of  oxidation  is  carried  on  by  the 
nitrifying  bacteria,  whereby  nitrates  are  ultimately  formed. 
These  are  eminently  adapted  for  use  by  plants,  and  so  the 
soil  is  rendered  continuously  capable  of  supporting  vege- 
tation. 

Nitrosomonas  and  Nitrosococcus  convert  ammonia  into 
nitrous  acid,  and  Nitrobacter  oxidizes  the  latter  to  form 
nitric  acid. 

These  genera  are  well  nigh  universal  in  the  soil.  They  do 
not  grow  on  the  ordinary  culture  media,  but  require  special 
solutions,  free  from  the  diffusive  albumins, — free,  indeed, 
from  organic  compounds  of  any  sort.  Their  supplies  of 
carbon  are  obtained  by  the  dissociation  of  carbon  dioxid. 
It  is  highly  noteworthy  that  they  are  thus  able  to  flourish 
without  food  more  complex  than  ammonia,  a  fact  which  is 
without  parallel  among  organisms  devoid  of  chlorophyl. 

Reduction  of  Nitrates. — A  considerable  number  of  bac- 
teria are  able  to  reduce  nitrogen  compounds  in  the  soil 
or  in  culture  media,  prepared  for  them,  into  ammonia. 
To  the  horticulturist  this  matter  is  of  much  interest. 
Winogradsky  *  has  described  specific  nitrifying  bacilli  which 
he  found  in  soil,  and  asserts  that  the  presence  of  ordinary 
bacteria  in  the  soil  causes  no  formation  of  nitrites  so  long 
as  the  special  bacilli  are  withheld. 

Reduction  of  nitrates  can  be  determined  experimentally  by 
the  use  of  a  nitrate  broth,  made  by  dissolving  in  1000  c.c.  of 
water  i  gram  of  peptone  and  0.2  gram  of  potassium  nitrate. 
The  ingredients  are  dissolved,  filtered,  then  filled  into  tubes, 

*  "Ann.  de  1'Inst.  Pasteur,"  1891;    "La  Semaine  medicale,"  1892. 


Combination  of  Nitrogen  75 

and  sterilized.  The  tubes  are  inoculated  and  the  results 
noted.  As  nitrites  and  ammonia  are,  however,  commonly 
present  in  the  air  and  are  taken  up  by  fluids,  it  is  always 
well  to  control  the  test  by  an  uninoculated  tube  tested 
with  the  reagents  in  the  same  manner  as  the  culture. 
Two  solutions  are  employed  *  for  testing  the  culture  : 


I.  Naphthylamin,  0.1  gram,          Boi1'    cofol>    **?•    *nd*d*  j5.6 
Distilled  water,  20.0  grams,  dilute   (1  :  16)    hydnc 


II.  Sulphanilic  acid,  0.5  gram. 

Hydric  acetate,  diluted,  150.0  c.c. 

Keep  the  solutions  in  glass-stoppered  bottles  and  mix  equal 
parts  for  use  at  the  time  of  employment. 

About  3  c.c.  of  the  culture  and  an  equal  quantity  of  the 
uninoculated  culture  fluid  are  placed  in  test-tubes  and  about 
2  c.c.  of  the  test  fluid  slowly  added  to  each.  The  develop- 
ment of  a  red  color  indicates  the  presence  of  nitrites,  the 
intensity  of  the  color  being  in  proportion  to  the  quantity 
of  nitrites  present.  If  a  very  slight  pinkish  or  reddish 
color  in  the  uninoculated  culture  fluid  and  a  deeper  red  in 
the  culture  develop,  it  shows  that  a  small  amount  of 
nitrites  was  already  present,  but  that  more  have  been  pro- 
duced by  the  growth  of  the  bacteria. 

The  presence  of  ammonia  in  either  fluid  is  easily  deter- 
mined by  the  immediate  development  of  a  yellow  color  or 
precipitate  when  a  few  drops  of  Nessler's  solution  f  are 
added. 

Failure  to  determine  either  ammonia  or  nitrites  may 
not  mean  that  the  nitrates  were  not  reduced,  but  that  they 
were  reduced  to  N.  It  is,  therefore,  necessary  to  test  the 
solutions  for  nitrates,  which  is  done  by  the  use  of  phenol- 
sulphonic  acid  and  sodium  hydroxid,  which  in  the  presence 
of  nitrates  give  a  yellow  color. 

Combination  of  Nitrogen.  —  Not  only  do  bacteria  de- 
stroy or  reduce  nitrogen  compounds,  but  some  of  them 
are  also  able  to  assimilate  nitrogen  from  the  air  and  so  com- 
bine it  as  to  be  useful  for  the  nourishment  of  vegetable 
and  animal  life.  The  most  interesting  organisms  of  this 

*  "Journal  of  the  American  Public  Health  Association,"  1888,  p.  92. 

t  Nessler's  solution  consists  of  potassium  iodid,  5  grams,  dissolved 
in  hot  water,  5  c.c.  Add  mercuric  chlorid,  2.5  grams,  dissolved  in  10 
cc.  of  water,  then  to  the  mixture  add  potassium  hydrate,  16  grams, 
dissolved  in  water,  40  c.c.,  and  dilute  the  whole  to  1000  c.c. 


76  Biology  of  Micro-organisms 

kind  are  found  upon  the  roots  of  the  leguminous  plants, 
peas,  clover,  etc.,  and  have  been  studied  by  Beyerinck.* 
It  seems  to  be  by  the  entrance  of  these  bacteria  into  their 
roots  that  the  plants  are  able  to  assimilate  nitrogen  from 
the  atmosphere  and  enrich  sterile  ground.  Every  agri- 
culturist knows  how  sterile  soil  is  improved  by  turning 
under  one  or  two  crops  of  clover  with  the  plough. 

Peptonization  of  Milk. — Numerous  bacteria  possess  the 
power  of  digesting — peptonizing — the  casein  of  milk.  The 
process  varies  with  different  bacteria,  some  digesting  the 
casein  without  any  apparent  change  in  the  milk,  some  pro- 
ducing coagulation,  some  gelatinization  of  the  fluid.  In 
some  cases  the  digestion  of  the  casein  is  so  complete  as  to 
transform  the  milk  into  a  transparent  watery  fluid. 

Milk  invariably  contains  large  numbers  of  bacteria,  that 
enter  it  from  the  dust  of  the  dairy,  many  of  them  pos- 
sessing this  power  and  ultimately  spoiling  the  milk.  In 
the  process  of  peptonization  the  milk  may  become  bitter, 
but  need  not  change  its  original  reaction. 

The  phenomena  of  coagulation  and  digestion  of  milk  can 
be  made  practical  use  of  to  aid  in  the  separation  of  simi- 
lar species  of  bacteria.  Thus,  the  colon  bacillus  coagulates 
milk,  but  the  typhoid  bacillus  does  not. 

Production  of  Disease. — Micro-organisms  that  produce 
disease  are  known  as  pathogenic;  those  that  do  not,  as  non- 
pathogenic.  Between  the  two  groups  there  is  no  sharp 
line  of  separation,  for  true  pathogens  may  be  cultivated 
under  such  adverse  conditions  that  their  virulence  may  be 
entirely  lost,  while  those  ordinarily  harmless  may  be  made 
virulent  by  certain  manipulations.  In  order  to  deter- 
mine that  a  micro-organism  is  possessed  of  pathogenic 
powers,  the  committee  of  bacteriologists  of  the  American 
Public  Health  Association f  recommends  that:  (i)  When  a 
given  form  grows  only  at  or  below  18°  to  20°  C.,  inoculation 
of  about  i  per  cent,  of  the  body-weight  with  a  liquid  culture 
seven  days  old  should  be  made  into  the  dorsal  lymph-sac 
of  a  frog.  (2)  When  a  species  grows  at  25°  C.  and  upward, 
an  inoculation  should  be  made  into  the  peritoneal  cavity 
of  the  most  susceptible  (in  general)  of  warm-blooded  animals 
— i.  e.,  the  mouse,  either  the  white  or  the  ordinary  house 
mouse.  The  inoculation  should  consist  of  about  i  per  cent. 

*  See  "Centralbl.  f.  Bakt.,"  etc.,  Bd.  vii,  p.  338. 
t  "Jour.  Amer.  Public  Health  Assoc.,"  Jan.,  1898. 


Production  of  Disease  77 

of  the  body- weight  of  the  mouse  of  a  four-  to  eight -hour 
standard  bouillon  culture,  or  a  broth  or  water  suspension 
of  one  platinum  loop  from  solid  cultures.  When  such 
intraperitoneal  injection  fails,  it  is  unlikely  that  other 
methods  of  inoculation  will  be  successful  in  causing  the 
death  of  the  mouse.  If  the  inoculations  of  the  frog  and 
mouse  both  prove  negative,  the  committee  think  it  un- 
necessary to  insist  upon  any  further  tests  of  pathogenesis 
as  being  requisite  for  work  in  species  differentiation. 

Production  of  Enzymes. — Some  of  these  have  already  been 
mentioned  as  causing  fermentation  and  putrefaction,  coagu- 
lating milk,  dissolving  gelatin,  etc.  There  are,  however, 
others  which  have  interesting  and  important  actions  upon 
both  animal  and  vegetable  substances. 

Knowledge  upon  the  subject  is  just  becoming  systema- 
tized, one  of  the  best  writings  being  by  Emmerich  and 
Low,*  who  observed  that  in  old  cultures  of  Bacillus  pyo- 
cyaneus  the  bacteria  become  transformed  into  a  gelatinous 
mass,  and  were  led  to  experiment  with  old  and  degener- 
ating cultures  condensed  to  ^  volume  in  a  vacuum  appa- 
ratus. The  bacteriolytic  powers  were  then  found  to  be 
much  increased,  and  they  were  subsequently  able  to  precipi- 
tate from  the  concentrated  culture  an  enzyme,  which  they 
called  pyocyanase.  The  authors  reach  the  rather  hasty  con- 
clusions that  the  cessation  of  growth  of  bacteria  in  cultures 
depends  upon  the  generation  of  enzymes;  that  the  enzymes 
destroy  the  dead  bacteria;  that  the  enzymes  will  kill  and 
dissolve  living  bacteria  and  destroy  toxins,  and,  therefore, 
are  useful  for  the  treatment  of  infectious  diseases;  and 
that  antitoxins  are  simply  accumulated  enzymes  which  the 
immunized  animals  have  received  during  treatment,  and 
which,  appearing  in  the  serum,  produce  the  effects  so  well 
known. 

It  is  probable  that  many  of  the  toxic  effects  of  bacteria 
and  their  cultures  depend  upon  enzymic  substances,  the 
nature  of  which  we  do  not  yet  understand. 

*  "  Zeitschrift  fur  Hygiene,"  1899. 


CHAPTER  III. 
INFECTION* 

INFECTION  is  the  successful  invasion  of  an  organism  by 
microparasites.  Unfortunately  custom  has  sanctioned  the 
use  of  the  word  in  other  and  sometimes  confusing  senses, 
thus,  a  table  or  knife  upon  which  micro-organisms  are  known 
to  be  or  are  even  supposed  to  be;  the  mouth  and  intestine, 
which  naturally  harbor  bacteria  of  various  forms,  or  a 
splinter  penetrating  the  skin  and  carrying  harmless  bacteria 
into  the  deeper  tissues,  are  all  said  by  the  surgeon  to  be 
"  infected,"  when,  in  fact,  it  would  be  more  correct  to  de- 
scribe them  as  infective. 

The  term  infection  should  imply  an  abnormal  state  result- 
ing from  the  deleterious  action  of  the  parasite  upon  the  host. 
The  colon  bacillus  is  a  harmless  commensal  of  the  intestine 
of  every  human  being,  and  of  most  of  the  lower  animals. 
The  intestine  is  not  "  infected,"  but  infested  with  it,  and  it  is 
only  when  abnormal  or  unnatural  conditions  arise  that 
infection  can  take  place.  This  form  of  association  of  certain 
bacteria  with  certain  parts  of  the  body  to  which  they  do  no 
harm,  but  into  which  they  may  rapidly  invade  when  appro- 
priate conditions  arise,  is  described  by  Adami  as  sub- 
infection.  The  possibility  of  infection  is  always  there, 
though  it  is  but  rarely  that  conditions  arise  under  which  it 
can  be  accomplished. 

There  are  two  inseparable  factors  to  be  considered  in  all 
infections :  the  organism  infecting  and  the  organism  infected. 
The  first  is  the  parasite,  the  second,  the  host.  Infectivity 
and  infectability  may  depend  upon  peculiarities  of  either 
parasite  or  host.  Organisms  that  have  lived  together  as 
commensals,  that  is,  in  a  state  of  neutral  relationship  for 
an  almost  indefinite  period,  may  suddenly  cease  their  cus- 
tomary association,  because  of  newly  acquired  power  of  in- 
vasion on  the  one  hand,  or  diminished  vital  resistance  on  the 

78 


Sources  of  Infection  79 

other,  and  infection  take  place  where  it  had  previously  been 
impossible. 

Bacteria  are  commonly  called  saprophytic  when  they 
live  in  nature  apart  from  other  living  organisms,  and  para- 
sitic when  they  live  in  or  upon  them.  Saprophytic  bacteria 
when  accidentally  transplanted  from  their  natural  environ- 
ment to  the  body  of  some  animal,  for  example,  may  or  may 
not  be  capable  of  continuing  life  under  the  new  conditions. 
In  the  greater  number  of  cases  they  die,  but  sometimes  the 
new  environment  seems  better  than  the  old,  and  they  mul- 
tiply rapidly,  invade  the  tissues  in  all  directions,  eliminate 
their  metabolic  products  into  the  juices,  and  occasion  vary- 
ing morbid  conditions. 

The  parasitic  bacteria  live  in  habitual  association  with 
higher  organisms.  Sometimes,  and  indeed  most  commonly, 
it  is  a  harmless  association,  like  that  of  certain  cocci  upon 
the  skin,  but  occasionally  it  results  in  the  destruction  of 
the  tissues  and  the  death  of  the  host,  as  in  tuberculosis, 
leprosy,  etc. 

The  group  of  pathogenic  organisms  has  no  well-defined 
limits,  for  it  is  frequently  observed  that  micro-organisms  well 
known  under  other  conditions,  and  not  known  to  have  been 
engaged  in  pathogenic  processes,  turn  up  unexpectedly  as 
the  cause  of  some  morbid  condition.  Indeed,  although  we 
are  acquainted  with  -u  large  number  of  organisms  that  have 
never  been  observed  in  connection  with  disease,  we  are  scarcely 
justified  in  concluding  that  they  are  incapable  of  producing 
injury  should  proper  conditions  arise. 

SOURCES  OF  INFECTION, 

The  sources  of  infection  may  be  exogenous  or  endogenous; 
that  is,  they  may  arise  through  the  admission  to  the  tissues 
of  micro-organisms  from  sources  entirely  apart  from  the 
individual  infected,  or  through  the  admission  of  some  of 
those  parasitic  and  usually  harmless  organisms  constantly 
associated  with  him. 

Exogenous  infections  arise  through  accidental  contact 
with  infective  objects  belonging  to  the  external  world. 

A  polluted  atmosphere  may  carry  into  the  respiratory 
passages  micro-organisms  capable  of  colonizing  there. 
Polluted  water  or  food  may  carry  into  the  intestine  micro- 
organisms whose  temporary  residence  may  entirely  change 
the  functional  and  structural  integrity  of  the  parts. 


8o  Infection 

Wounds  inflicted  by  the  teeth  of  animals,  by  weapons,  by 
implements,  or  by  objects  of  various  kinds,  carry  into  the 
tissues  micro-organisms  whose  operations,  local  or  general, 
may  variously  affect  the  organism  to  its  detriment. 

Contact  with  unclean  objects  of  various  kinds — spoons, 
knives,  cups,  blow-pipes,  catheters,  syringes,  dental  instru- 
ments, etc. — may  serve  to  transfer  disease-producing  organ- 
isms from  one  person  to  another  who  might  otherwise  never 
come  in  contact  with  them. 

Suctorial  insects  seem  occasionally  to  act  as  the  medium 
by  which  micro-organisms  withdrawn  in  blood  from  one 
person  may  be  introduced  into  other  persons  so  that  they 
become  infected.  The  flea  thus  brings  about  the  spread  of 
plague;  the  mosquito,  of  malaria;  the  tsetse  fly,  of  trypano- 
somiasis;  the  tick,  of  relapsing  fever. 

Fomites,  or  objects  made  infective  through  contact  with 
individuals  suffering  from  smallpox,  scarlatina,  and  other 
contagious  or  actively  infectious  diseases,  become  the  means 
through  which  the  specific  micro-organisms  may  be  con- 
veyed to  the  well  with  resulting  infection. 

Endogenous  infections  arise  through  the  activity  of 
micro-organisms  habitual  to  the  body.  It  indicates  a 
morbid  condition  of  the  body  by  which  the  defensive  mechan- 
isms are  disturbed,  so  that  organisms  harmless  under  normal 
conditions  become  invasive. 

All  normal  animals  are  born  free  of  parasitic  micro-organ- 
isms, but  it  is  impossible  for  them  to  remain  so  because  of 
the  universal  distribution  of  micro-organismal  life.  The 
air,  the  water,  the  soil,  and  the  food,  as  well  as  the  associates 
of  the  young  animal,  all  act  as  means  by  which  micro-organ- 
isms, and  especially  bacteria,  are  brought  to  the  surface  and 
cavities  of  its  body,  and  but  a  short  time  elapses  after  birth 
before  it  harbors  the  customary  commensal  and  parasitic 
forms. 

BACTERIAL  TENANTS  OF  THE  NORMAL  HUMAN  BODY. 

The    skin    and    adjacent    mucous    membranes.      The 

slightly  moist  warm  surface  of  the  skin  is  well  adapted  to 
bacterial  life,  and  its  unavoidable  contact  with  surrounding 
objects  determines  that  a  variety  of  organisms  shall 
adhere  to  it.  Of  these,  we  can  differentiate  between  forms 
whose  presence  is  unexpected  and  temporary ;  others  whose 


Bacterial  Tenants  of  the  Normal  Human  Body    81 

presence  may  be  expected;  and  still  others  whose  presence 
is  invariable. 

Elaborate  investigations  upon  the  bacterial  flora  of  the 
skin  have  been  made  by  Unna*;  Mittman,f  who  studied  the 
finger-nails,  under  which  he  found  no  less  than  seventy- 
eight  different  species ;  Maggiora,  J  who  isolated  twenty-nine 
forms  from  the  skin  of  the  foot;  and  Preindelsberger, §  who 
found  eighty  species  of  bacteria  on  the  hands.  Undoubtedly 
many  of  these  organisms  were  accidentally  present,  and  were 
at  least  only  semi-parasitic.  Not  a  few  were  met  but  once 
and  were  in  no  sense  bacteria  of  the  skin.  The  skin  may 
also  be  temporarily  contaminated  with  bacteria  from  other 
portions  of  the  patient's  body,  as,  for  instance,  from  his 
intestine ;  thus  Winslow  ||  has  found  the  colon  bacillus  upon 
the  hands  of  ten  out  of  one  hundred  and  eleven  persons 
examined.  Wigura**  also  examined  the  hands  of  forty 
persons  in  hospitals,  finding  tubercle  bacilli  in  two  out  of 
ten  persons  from  phthisical  wards  in  hospitals,  colon  bacilli 
six  times  and  typhoid  bacilli  once  on  the  hands  of  nine 
attendants  in  the  typhoid  wards.  He  found  streptococci 
and  staphylococci  many  times.  Welchff  and  Robb  and 
GhriskeyJ  J  seem  to  have  been  the  first  to  make  a  clear  dif- 
ferentiation between  the  accidentally  present  bacteria  and 
the  permanently  parasitic  organisms  of  the  skin,  and  to  show 
that  certain  cocci,  producing  white  and  yellow  colonies  upon 
agar-agar,  were  invariable  in  occurrence  and  penetrated  to 
the  lowest  epidermal  layers. 

These  cocci,  of  which  Welch  describes  the  most  common 
as  Staphylococcus  epidermidis  albus,  are  universally  and 

*  "  Monatshefte  fiir  prakt.  Dermatol.,"  vii,  1888,  p.  817  ;vm,  1889, 
pp.  293,  562;  ix,  1889,  p.  49;  x,  1890,  p.  485;  xi,  1890,  p.  471;  xn, 
1891,  p.  249. 

f'Archiv.  f.  path.  Anat.  u.  Phys.  u.  f.  klin.  Med.,"  cxm,  1888,  p. 
203. 

f'Giornale  della  R.  Societd  d'Igiena,"   1889,  Fasc.  5,  p.  335. 
§"  Samml.  Medic.  Schriften,"  herausg.  von  der  "  Wiener  klin.  Woch- 
enschrift,"    xxii,   Wien,    1891;    "Rev.   Jahresbericht  iiber   die   Fort- 
schritten  in  der   Lehre   von  den  pathogenen  Mikroorganismen,"  vii, 
1891,  p.  619. 

||  "  Jour.  Med.  Research,"  vol.  x,  p.  463. 
**  "Wratsch,"  1895,  No.  14. 

ft  "Transactions  of  the  Congress  of  American  Physicians  and  Sur- 
geons," n,  1891,  p.  1. 

tt  "  Bulletin  of  the  Johns  Hopkins  Hospital,"  m,  1892,  p.  37, 
6 


82  Infection 

invariably  present  upon  the  human  skin,  and  must  be  re- 
garded as  habitual  parasites. 

Where  the  skin  is  peculiar  in  its  moisture  and  greasiness, 
however,  additional  forms  are  found.  Thus,  in  preputial 
smegma,  in  the  axillae,  and  sometimes  about  the  lips  and 
nostrils,  a  bacillary  organism,  Bacillus  smegmatis,  is  invari- 
able, and  the  recent  work  by  Schaudinn  and  Hoffman*  has 
shown  that  the  skin  of  the  genitalia  harbors  a  spiral  organism 
which  they  call  Spirochseta  refringens. 

In  the  external  auditory  meatus  a  coccus,  Micrococcus 
cereus  flavus,  is  almost  always  to  be  found  in  the  waxy 
secretion. 

Upon  the  conjunctiva  as  many  accidental  organisms  may 
be  found  as  shall  have  been  caught  by  its  moist  surface, 
though  the  researches  of  Hildebrand  and  Bernheim  and 
others  seem  to  show  that  the  tears  have  some  antiseptic 
power  and  prevent  the  organisms  from  growing,  so  that  in 
health  there  are  very  few  permanent  residents  of  the  sac, 
certain  cocci  seeming  to  be  the  only  constant  forms. 

The  mouth  has  been  carefully  studied  bacteriologically 
by  Miller,  f  who  found  six  organisms — Leptothrix  innomi- 
nata,  Bacillus  buccalis  maximus,  Leptothrix  buccalis 
maxima,  lodococcus  vaginatus,  Spirillum  sputigenum  and 
Spirochaeta  dentinum  (denticola) — in  every  mouth.  Prac- 
tically the  same  conclusions  were  reached  by  Vincentini.  J 
These  organisms  are  peculiar  in  that  they  will  not  grow  in 
artificial  culture.  In  addition  to  this  permanent  flora, 
Miller  cultivated  fifty- two  other  species,  some  of  which  were 
harmless,  some  well-known  pathogens. 

From  the  mouth  these  organisms  may  be  traced  into  the 
pharynx  and  esophagus. 

In  studying  the  micro-organisms  of  dental  caries  Goodby§ 
found  a  large  number  of  organisms  which  he  divided  into 
three  groups:  A.  Those  that  produce  acids,  including 
Streptococcus  brevis,  Bacillus  necrodentalis  (Goodby), 
Sarcina  alba,  Sarcina  lutea,  Sarcina  aurantiaca,  Staphylo- 
coccus  pyogenes  aureus,  and  Staphylococcus  pyogenes  sali- 
varius  (Biondi).  B.  Those  that  liquefy  blood-serum:  Ba- 

*  "Deutsche  med.  Woch.,"  May  4,  1905. 
t  "Micro-organisms  of  the  Human  Mouth,"  Phila.,  1890. 
%  "Bacteria  of  the  Sputa  and  Cryptogamic  Flora  of  the  Mouth," 
London,  1897. 

§  Transactions  of  the  Odontological  Society,  June,  1899. 


Bacteria  of  the  Digestive  Apparatus  83 

cillus  mesentericus  rubra,  B.  mesentericus  vulgatus,  B. 
mesentericus  fuscus,  Bacillus  fuscus,  a  yellow  bacillus, 
probably  B.  gingival  pyogenes  (Miller),  and  Bacillus  lique- 
facium  motilis.  C.  Those  that  produce  pigment,  including 
the  same  organisms  as  group  B.  In  carious  dentine  two 
organisms,  Streptococcus  brevis  and  Bacillus  necrodentalis, 
were  invariably  present. 

The  extinction  of  the  great  number  of  bacteria  entering 
the  mouth  is  referred  by  most  bacteriologists  to  a  bac- 
tericidal action  of  the  saliva. 

The  stomach  seems  to  retain  very  few  of  the  many 
bacteria  that  must  enter  it,  its  persistently  acid  contents 
being  inimical  to  their  development.  Certain  sarcina, 
especially  Sarcina  ventriculi,  may  be  found  without  any 
considerable  departure  from  the  normal  state.  In  carcinoma 
and  other  forms  of  pyloric  obstruction  with  dilatation,  the 
bacterial  flora  increases,  and  in  achlorhydria  micro-organ- 
isms of  fermentation  make  their  appearance.  They  are, 
however,  accidental  and  not  permanent  tenants  of  the  organ. 

In  carcinoma  of  the  stomach  a  bacillus,  probably  one  of 
the  lactic  acid  groups,  early  makes  its  appearance  and  is  of 
some  diagnostic  importance.  It  is  called  after  its  discoverer 
the  Oppler-Boas  bacillus,*  also  on  account  of  angulations 
found  in  its  threads,  Bacillus  geniculatus.  It  is  a  large 
bacillus,  tending  to  form  long  threads  easily  seen  without  an 
oil-immersion  lens.  It  is  probably  non-motile,  does  not 
form  spores,  stains  by  Gram's  method,  and  is  said  by  Emory  f 
to  divide  longitudinally  as  well  as  transversely.  This,  as 
he  says,  will,  if  proved  to  be  correct,  be  a  most  important 
means  of  identifying  the  species.  Cultures  are  easily  made 
in  media  acidified  with  lactic  acid. 

The  intestine  receives  such  micro-organisms  as  have 
survived  whatever  destructive  influences  the  gastric  juices 
may  have  exerted,  and  its  alkaline  contents,  rich  in  proteins 
and  carbohydrates  in  solution,  are  eminently  appropriate  for 
bacterial  life.  The  flora  of  the  intestine  is,  therefore,  in- 
creased in  number  and  variety  of  organisms  as  we  descend 
from  its  beginning  to  its  end.  In  the  small  intestine  there 
may  be  no  bacteria  in  the  upper  part  of  the  jejunum,  but 
in  most  cases  Bacillus  lactic  aerogenes  and  bacilli  of  the 
colon  groups  are  found.  These  increase  in  number  as  the 

*"  Deutsche  tned.  Wochenschrift,"  1905,  No.  5. 
t  "Bacteriology  and  Hematology,"  p.   114. 


84  Infection 

iliocecal  valve  is  reached.  The  cecum  shows  large  num- 
bers of  colon  bacilli.  The  rectum  contains,  in  addition, 
many  putrefactive  organisms,  such  as  Bacillus  putrificus, 
Bacillus  proteus  vulgaris,  members  of  the  Bacillus  subtilis 
group,  and  acid-producing  organisms,  such  as  Bacillus 
acidophilus. 

An  interesting  and  thorough  study  of  these  organisms 
of  the  bowel  and  their  distribution  has  been  made  by  Kohl- 


Fig.  17.— Sarcina  ventriculi  (Migula). 

brugge.*  The  total  number  of  permanent  residents  is  not 
known.  During  infancy  the  predominating  organism  seems 
to  be  Bacillus  lactis  aerogenes;  during  adult  life,  Bacillus 
coli.  Streptococci,  especially  Streptococcus  coli  gracilis, 
are  also  very  common,  if  not  invariable,  inhabitants  of  the 
intestine.  The  total  bacteria  that  finally  appear  in  the 
feces,  according  to  the  studies  of  Strasburgerf  and  Steele,J 
may  reach  the  enormous  figure  of  38  per  cent,  of  the  total 
bulk. 

MacNeal,  Latzer,  and  Kerr,§  in  an  elaborate  work  upon  the 
"  Fecal  Bacteria  of  Healthy  Men,"  found  that  they  furnished 
46.3  per  cent,  of  the  total  fecal  nitrogen. 

:  "Centralbl.  f.  Bakt.,"  etc.,  Bd.  xxx,  1901,  pp.  10  and  70. 
f  "Zeitschrift  fur  klin.  med.,"   1902,  xuv,  5  and  6;    1903,  XLVIII, 
5  and  6. 

J  "Jour.  Amer.  Med.  Assoc.,"  Aug.  24,  1907,  p.  647. 

$  "Journal  of  Infectious  Diseases,"  1909,  vi,  pp.  132,  571. 


Bacteria  of  the  Digestive  Apparatus  85 

Rettger  *  found  the  Bacillus  enteritidis  sporogenes  regu- 
larly present  in  the  human  feces  and  believes  it  to  be  respon- 
sible for  some  of  the  putrefactive  processes  that  occur  there. 

The  vagina,  on  account  of  its  acid  secretions,  harbors 
but  few  bacteria.  In  a  study  of  the  vaginal  secretions  of 
40  pregnant  women  who  had  not  been  subjected  to  digital 
examinations,  douches,  or  baths,  Bergholm  t  found  but 
few  organisms  of  limited  variety. 

The  uterus  harbors  no  bacteria  in  health,  and  but  few  in 
disease.  The  intervening  acidity  of  the  vagina  makes  it 
difficult  for  bacteria  from  the  surface  to  penetrate  so  deeply, 
and  tha  tenacious  alkaline  mucus  of  the  cervix  is  an  addi- 
tional barrier  to  their  progress.  Careful  studies  of  the 
bacteriology  of  the  uterine  secretions  have  been  made  by 
Gottschalk  and  Immerwahr  J  and  Doderlein  and  Win- 
terintz.  § 

The  urethra  harbors  a  few  cocci  which  enter  the  meatus 
from  the  surface  and  remain  local  in  distribution. 

The  normal  bladder  is  free  from  bacteria. 

The  nose  constantly  receives  enormous  numbers  of  bac- 
teria in  the  dust  of  the  inspired  atmosphere.  These  organ- 
isms are  too  numerous  and  too  various  to  enumerate,  and 
might,  indeed,  comprehend  the  entire  bacterial  flora.  But 
in  spite  of  the  large  numbers  of  organisms  received,  the  nose 
retains  scarcely  any,  its  mucous  membranes  seeming  to  be 
provided  with  means  of  disposing  of  the  organisms.  Among 
those  best  able  to  withstand  the  destructive  influences,  and, 
therefore,  most  apt  to  be  found  in  the  deeper  passages,  are 
the  pseudodiphtheria  bacillus,  streptococci,  pneumococci, 
staphylococci,  Bacillus  pneumoniae  (Friedlander) ,  Bacillus 
subtilis  and  sarcina.  A  complete  review  of  the  subject  with 
references  to  the  literature  has  been  made  by  Hasslauer.|| 

The  larynx  and  trachea  contain  very  few  bacteria  and 
probably  have  no  permanent  parasitic  flora. 

The  lungs  harbor  no  bacteria.  A  few  micro-organisms 
doubtless  reach  them  in  the  inspired  air,  but  the  defensive 
mechanisms  soon  dispose  of  them. 

*  "Jour,  of  Biological  Chemistry,"  n,  i  and  2,  Aug.,  1906,  p.  71. 

t  "Archiv  f.  Gynak.,"  Bd.  LXIV,  Heft.  3. 

J  Ibid.,  1896,  Bd.  L,  Heft  3. 

§  Hegar's  "  Beitrage  fur  Geburtshiilfe  und  Gynakologie,"  Bd.  in, 
Heft.  2. 

||  "Centralbl.  f.  Bakt.  u.  Parasitenk.  I.  abt.  Referata.,"  Bd.  xxxvn, 
Nos.  1-3,  p.  i,  and  Nos.  4-6,  p.  97. 


86  Infection 


AVENUES  OF  INFECTION. 

The  skin  seems  to  form  an  effectual  barrier  against  the 
entrance  of  bacteria  into  the  deeper  tissues.  A  few  higher 
fungi — Trycophyton,  Microsporon,  Achorion,  etc. — seem 
able  to  establish  themselves  in  the  superficial  layers  of  the 
cells,  invade  the  hair-follicles,  and  so  reach  the  deeper 
layers,  where  morbid  changes  are  produced.  The  minute 
size  of  the  bacteria  makes  it  possible  for  them  to  enter 
through  lesions  too  small  to  be  noticed.  Garre  applied  a 
pure  culture  of  Staphylococcus  pyogenes  aureus  to  the  skin 
of  his  forearm,  and  found  that  furuncles  developed  in  four 
days,  though  the  skin  was  supposed  to  be  uninjured.  Bock- 
hart  moistened  his  skin  with  a  suspension  of  the  same  organ- 
ism, gently  scratched  it  with  his  finger-nail,  and  suffered 
from  a  furuncle  some  days  later. 

The  greater  number  of  surgical  infections  result  from  the 
entrance  of  bacteria  through  lesions  of  the  skin.  It  makes 
but  little  difference  to  what  depth  the  lesion  extends,— 
abrasions,  punctures,  lacerations,  incisions, — the  protective 
covering  is  gone  and  the  infecting  organisms  find  themselves 
in  the  tissues,  surrounded  by  the  tissue  lymph,  under  con- 
ditions appropriate  for  growth  and  multiplication,  provided 
no  inhibiting  or  destructive  mechanism  be  called  into  action. 

The  digestive  apparatus  is  the  portal  through  which 
many  infections  take  place.  The  Bacillus  diphtherise, 
finding  its  way  to  the  pharynx,  speedily  establishes  itself 
upon  the  surface,  producing  pseudomembranous  inflam- 
mation there.  Typhoid  bacilli,  dysentery  bacilli,  cholera 
spirilla  and  related  organisms,  finding  their  way  to  the 
intestine,  where  the  vital  conditions  are  appropriate,  take 
up  temporary  residence  there,  to  the  inconvenience  of  the 
host,  who  suffers  from  the  respective  infections. 

Various  organisms  pass  from  the  pharynx  to  the  tonsils 
and  so  to  the  lymph-nodes  and  deeper  tissues  of  the  neck, 
where  their  first  operations  may  be  observed. 

It  is  supposed  by  some  pathologists  that  the  digestive 
tract  is  a  constant  menace  to  health  in  that  it  regularly 
admits  bacteria,  through  the  lacteals,  and  perhaps  through 
its  capillaries,  to  the  blood,  where  under  slightly  abnormal 
conditions  they  might  do  harm.  According  to  Adami,* 

*  "Jour,  of  the  American  Medical  Association,"  Dec.  16  and  23, 
1899,  vol.  xxxm,  Nov.  25  and  26. 


The  Avenues  of  Infection  87 

the  intestine  is  responsible  -  for  a  condition  of  sub-infection 
depending  upon  the  constant  entrance  of  colon  bacilla  into 
the  blood.  He  finds  the  colon  bacillus  in  the  blood,  and 
traces  it  to  the  liver,  where  its  final  dissolution  takes  place 
in  the  fine  dumbbell-like  granules  enclosed  in  the  cells,. 
Nicholls*  confirms  Adami  by  finding  similar  dumbbell  or 
diplococcoid  bodies  in  the  epithelial  denuded  tissues  of  the 
mesentery  of  normal  animals. 

Nicholas  and  Descosf  and  RavenelJ  fed  fasting  dogs  upon 
a  soup  containing  quantities  of  tubercle  bacilli,  killed  them 
three  hours  later,  and  examined  the  contents  of  the  thoracic 
duct,  where  tubercle  bacilli,  some  alive  and  some  dead,  were 
found  in  large  numbers,  van  Steenberghe  and  Grysez§ 
found  that  carbon  particles  readily  passed  through  the 
intestinal  mucosa,  entered  the  lymphatics,  were  thrown 
into  the  venous  circulation,  and  so  carried  to  the  lung,  where 
anthracosis  was  produced. 

In  a  subsequent  paper  ||  they  believe  that  they  have  demon- 
strated that  the  tubercle  bacillus  like  the  carbon  particles 
may  also  pass  through  the  normal  intestinal  wall,  and  follow 
the  same  course  to  the  lungs.  They  believe  that  pulmonary 
tuberculosis  thus  depends  upon  ingested  and  not  inhaled 
micro-organisms.  At  my  suggestion  Montgomery**  en- 
deavored to  repeat  the  work  of  van  Steenberghe  and  Grysez 
at  the  Henry  Phipps  Institute,  Philadelphia,  but  though 
many  attempts  were  made  by  various  methods,  no  carbon 
particles  were  transported  from  the  alimentary  to  the  pul- 
monary tissues. 

But  there  are  enough  experiments  recorded  to  make  it 
probable  that  the  wall  of  the  intestine  is  permeable  to  bacteria, 
and  that  in  small  numbers  they  constantly  enter  the  blood  of 
healthy  animals,  to  be  disposed  of  by  mechanisms  yet  to  be 
described. 

Many  of  the  bacteria  penetrating  the  intestine  must  be 
retained  in  the  lymph  nodes;  others,  as  in  the  experiment 
with  the  tubercle  bacilli,  meet  destruction  before  they  reach 
the  blood;  the  remainder  must  reach  the  blood  alive. 

*  "Jour.  Med.  Research.,"  vol.  xi,  No.  2. 
t  "Jour,  de  Phys.  et  Path,  gen.,"  1902,  iv,  910-912. 
I  "Jour.  Med.  Research."  x,  p.  460,  1904. 

§  "Ann.  de  1'Inst.  Pasteur,"  Tome  xix,  No.  12,  p.  787,  Dec.  25,  1905. 
||  Ibid.,  1910,  xxiv,  316. 
**  "Jour,  of  Med.  Research,"  Aug.,  1910,  vol.  xxm,  No.  i. 


88  Infection 

The  presence  of  colon  bacilli  in  the  greater  number  of  the 
organs  shortly  after  death  has  led  some  pathologists  to 
assume  that  they  readily  pass  through  the  intestinal  walls 
during  the  death  agony,  but  although  experiments  have  been 
made  to  prove  and  to  disprove  it,  the  matter  is  still  con- 
troversial. Undoubtedly  in  the  final  dissolution  some 
change  takes  place  in  the  constitution  of  the  individual  by 
which  general  invasion  by  bacteria  is  made  more  easy  than 
under  normal  conditions. 

The  respiratory  apparatus  affords  admission  to  a  few 
micro-organisms  whose  activities  seem  more  easily  carried 
on  there  than  elsewhere.  Although  it  is  still  controversial 
whether  the  inhalation  of  tubercle  bacilli  is  as  frequent  a 
mode  of  conveying  that  organism  into  the  body  as  was  once 
supposed,  it  cannot  be  denied  that  its  inhalation  will  account 
for  the  far  greater  frequency  with  which  tuberculosis  affects 
the  lungs  than  other  organs  of  the  body. 

Pneumonia,  caused  in  an  immense  majority  of  cases  by 
the  pneumococcus  of  Fraenkel  and  Weichselbaum,  probably 
results  from  the  entrance  of  the  organism  into  the  respira- 
tory tissues  directly. 

The  entrance  of  the  unknown  infectious  agents  causing 
measles,  German  measles,  smallpox,  and  scarlatina,  can  best 
be  accounted  for  by  supposing  that  they  are  inhaled  into 
the  lungs  and  thus  enter  the  blood. 

The  genital  apparatus  is  the  portal  of  entry  of  micro- 
organisms whose  early  or  chief  operations  are  local.  Among 
these  are  the  gonococcus,  which  causes  urethritis,  vaginitis, 
balanitis,  posthitis,  endometritis,  orchitis,  salpingitis,  vesicu- 
litis,  cystitis,  oophoritis,  sometimes  peritonitis,  and  rarely 
endocarditis;  the  bacillus  of  Ducrey,  that  causes  the  chan- 
croid or  soft  sore;  and  the  treponema  of  syphilis.  In  more 
rare  cases  other  organisms,  such  as  the  common  cocci  of 
suppuration  and  the  tubercle  bacillus,  may  also  be  trans- 
mitted from  individual  to  individual  by  sexual  contact. 

The  placenta  usually  forms  a  barrier  through  which 
infectious  agents  find  their  way  with  difficulty.  A  study 
of  this  subject  by  Neelow*  shows  that  the  non-pathogenic 
organisms  do  not  pass  from  the  mother  through  the  placenta 
to  the  fetus.  Some  pathogenic  micro-organisms,  however, 
readily  pass  through,  and  a  few  diseases,  such  as  syphilis, 
are  well  known  in  the  congenital  form.  Pregnant  women 
*  "Centralbl.  f.  Bakt.,"  etc.,  I.  Abt.  Bd.  xxxi,  Orig.,  Aug.,  1902,  p.  691. 


Pathogenesis  89 

suffering  from  smallpox  may  be  delivered  of  infants  with 
marks  indicative  of  prenatal  disease.  Some  common 
infectious  agents,  such  as  the  tubercle  bacillus,  seem  to  infect 
unborn  animals  with  difficulty.  The  frequency  of  antenatal 
tuberculous  infection  is,  however,  somewhat  controversial 
at  present,  Baumgarten  having  reached  the  opinion,  exactly 
the  opposite  of  what  is  commonly  believed,  that  most  children 
are  subject  to  antenatal  infection,  though  the  bacilli  sub- 
sequently develop  and  cause  disease  in  only  a  few  of  them. 


PATHOGENESIS. 

This  subject  can  be  understood  only  through  a  broad 
knowledge  of  the  metabolic  products  of  micro-organisms. 
In  general  it  may  be  said  that  the  ability  of  micro-organisms 
to  do  harm  depends  upon  the  injurious  nature  of  their 
products.  This  alone,  however,  will  not  explain  the  phe- 
nomena of  infection,  for  in  many  cases  the  intoxication  is 
subsidiary  in  importance  to  the  invasive  power  of  the  micro- 
organisms. Some  bacteria  having  but  limited  toxic  powers 
possess  extraordinary  powers  of  invasion,  as  Bacillus  an- 
thracis,  and  the  intoxication  becomes  important  only  after  the 
organisms  have  penetrated  to  all  the  tissues  of  the  body. 
Others,  with  more  active  toxic  properties,  have  but  limited 
invasive  powers,  and  a  few  organisms,  growing  with  difficulty 
in  some  insignificant  focus,  excite  actively  destructive 
reactions  in  the  tissues  with  which  they  come  into  contact. 
Still  others,  with  limited  invasive  powers,  eliminate  active 
toxic  substances,  soluble  in  nature,  that  enter  the  circulation 
and  act  upon  cells  remote  from  the  bacteria  themselves,  as 
in  diphtheria  and  tetanus. 

The  invasive  power,  of  the  organisms  depends  upon  their 
ability  to  overcome  the  body  defenses.  This  may  indicate 
activity  of  the  infecting  organism,  or  weakness  of  the  defen- 
sive mechanism.  The  relation  of  these  factors  is  exceedingly 
complex,  only  partly  understood,  and  will  be  fully  discussed 
in  the  chapter  upon  Immunity. 

For  convenience  toxins  may  be  described  as  intracellular 
or  insoluble,  and  extracellular  or  soluble. 

The  intracellular  toxins.  These  products  are  but  little 
known  and  have  only  recently  begun  to  attract  attention. 
Their  insoluble  nature  makes  it  difficult  to  isolate  them,  and 
determines  the  limitations  of  their  activity.  Until  the  in- 


go  Infection 

vestigations  of  Vaughan,  Cooley  and  Gelston,*  and  later 
Vaughan  and  his  associates,  Detweiler,f  Wheeler, J  Leach, § 
Marshall  and  Gelston,  ||  Gelston,**  J.  V.  Vaughan,ft 
Wheeler, ft  Leach, §§  Mclntyre,||  ||  and  others,  it  seemed  re- 
markable that  micro-organisms  whose  filtered  cultures  con- 
tained little  demonstrable  toxic  substance  are  sometimes 
able  to  produce  active  pathogenic  effects.  By  means  of 
special  apparatus  in  which  the  micro-organisms  could  be 
cultivated  in  enormous  quantities,  and  the  disintegration  of 
the  micro-organismal  masses  secured  by  subjecting  them 
to  high  temperatures,  to  the  action  of  mineral  acids  or 
autolysis,  it  was  discovered  that  the  colon  bacilli,  typhoid 
bacilli,  and  many  supposedly  harmless  bacteria,  contain 
intensely  active  toxic  substances.  In  all  probability  some 
of  the  toxic  substances  produced  by  such  means  are  artefacts, 
but  enough  work  has  been  done  to  prove  that  insoluble  toxic 
substances  are  present  in  such  organisms,  and  the  toxic 
substances  obtained  by  the  comminution  of  culture  masses 
made  solid  and  brittle  by  exposure  to  liquid  air,  as  suggested 
by  Macfadyen  and  Rowland;  the  autolytic  digestion  of  bac- 
teria washed  free  of  their  culture  fluids  and  suspended  in 
physiological  salt  solution,  and  the  dissolution  of  bacteria 
by  bacteriolytic  animal  juices  clearly  prove  that  endotoxins 
exist. 

It  seems  probable  that  there  is  considerable  difference  in 
the  readiness  with  which  these  intracellular  toxic  substances 
are  given  up  by  the  bacteria.  From  some  they  seem  never 
to  be  set  free  in  the  bodies  of  animals  into  which  the  bacteria 
are  injected;  thus,  Bacillus  prodigiosus  is  harmless  for 
animals,  no  matter  what  quantity  is  injected,  yet  active 
toxic  substances  can  be  extracted  from  the  bodies  of  these 
organisms  by  appropriate  chemical  means.  From  others 
they  are  given  off  in  small  quantities  either  during  the  life 
of  the  organism,  or  at  the  moment  of  death  and  dissolution, 
as  in  the  case  of  the  typhoid  bacillus  and  streptococci,  whose 
filtered  cultures  are  almost  harmless,  though  both  organisms 
are  pathogenic. 

*  "Journal  of  the  American  Medical  Association,"  Feb.  23,  1901; 
"Trans.  Assoc.  Amer.  Phys.,"  1901,  "American  Medicine,"  May, 
1901. 

t  "Trans.  Assoc.  Amer.  Phys.,"  1902.  J  Ibid. 

§  Ibid.  ||  Ibid.  **  Ibid.  ft  Ibid. 

|J  "Jour.  Amer.  Med.  Assoc.,"  1904,  xui,  p.  1000. 
§§  Ibid.,  p.  1003.  llll  Ibid.,  p.  1073 


Pathogenesis  91 

The  intracellular  toxins  are  limited  in  action  by  the 
distribution  of  the  bacteria  producing  them.  When  these 
organisms  are  but  slightly  invasive,  more  or  less  local  reaction 
is  produced ;  when  they  are  actively  invasive,  general  reac- 
tions of  varying  intensity  result. 

The  extracellular  toxins,  of  which  those  of  Bacillus 
tetani  and  Bacillus  diphtheriae  can  be  taken  as  types,  have 
been  known  since  the  early  work  of  Brieger  and  Fraenkel 
and  Roux  and  Yersin.  They  seem  to  be  excretions  of  the 
bacteria,  not  retained  in  the  cells,  but  eliminated  from  them 
as  rapidly  as  they  are  formed.  Thus,  in  appropriate  bouillon 
cultures  of  the  diphtheria  bacillus,  the  toxin  is  present  in 
large  quantity  and  is  highly  virulent,  but  if  the  fluid  be 
removed  from  the  bacteria  by  porcelain  filtration  and  the 
remaining  bacilli  carefully  washed,  their  bodies  are  found  to 
be  devoid  of  toxic  powers.  The  poison  is  most  concentrated 
where  its  diffusion  is  most  restricted,  thus,  agar-agar  cultures 
of  the  tetanus  bacillus  are  much  more  toxic  than  bouillon 
cultures  because  the  soluble  principle  readily  diffuses  through 
the  fluid,  but  is  held  by  the  less  diffusible  agar-agar. 

The  soluble  toxin  is  but  one  of  numerous  metabolic  prod- 
ucts of  the  bacteria.  Thus  in  culture  filtrates  of  the 
tetanus  bacillus  there  are  at  least  two  very  different  active 
substances,  the  tetano-spasmin  that  acts  upon  the  nervous 
system  with  convulsive  effect,  and  the  tetano-lysin  that  is 
solvent  for  erythrocytes. 

In  all  probability  all  of  the  culture  filtrates  of  bacteria 
are  highly  complex  because  of  the  addition  of  the  various 
metabolic  products — toxins,  lysins,  enzymes,  pigments,  acids, 
etc. — of  the  bacteria,  as  well  as  because  of  changes  produced 
in  the  medium  by  the  abstraction  of  those  molecular  con- 
stituents upon  wh'ich  the  bacteria  have  fed.  This  com- 
plexity makes  it  difficult  to  accurately  study  the  toxins, 
which  we  scarcely  know  apart  from  their  associated  products. 

The  chemic  nature  of  the  toxins  differs.  Undoubtedly 
some  are  tox-albumins,  but  others  are  of  different  composi- 
tion and  fail  to  give  the  reactions  belonging  to  the  compounds 
of  this  group. 

The  variations  observed  in  toxicogenesis  under  experi- 
mental conditions  in  the  test-tube  indicate  that  similar 
variations  occur  in  the  bodies  of  animals,  and  a  few  experi- 
ments conducted  with  slight  variations  in  the  composition 
and  reaction  of  the  media  in  which  the  bacteria  grow  will 


92  Infection 

suffice  to  show  that  the  exact  effect  of  toxicogenic  bacteria 
in  the  bodies  of  different  animals  cannot  always  be  accurately 
prejudged. 

The  physiologic  and  pathogenic  action  of  the  extracellular 
soluble  toxins  differs  from  that  of  the  intracellular  and  dif- 
ficultly soluble  toxins  in  that  it  is  more  easily  diffused 
throughout  the  animal  juices,  and  that  its  diffusion  is  inde- 
pendent of  the  invasiveness  of  the  bacteria,  so  that  a  few 
organisms  growing  at  some  focus  of  unimportant  magnitude, 
and  causing  but  little  local  manifestation,  may  be  able  to 
produce  a  profound  impression  upon  remote  organs.  This 
is  best  exemplified  in  the  case  of  the  Bacillus  tetani,  which, 
finding  its  way  into  the  tissues  under  proper  conditions, 
produces  scarcely  any  local  reaction, — indeed,  the  lesion  may 
be  undiscoverable, — yet  may  cause  the  death  of  the  animal 
through  the  intensity  of  its  action  upon  the  central  nervous 
system. 

SPECIFIC  ACTION  OF  TOXINS. 

The  metabolic  products  of  the  greater  number  of  injurious 
bacteria  are  characterized  by  irritative  action  upon  those 
body  cells  with  which  they  come  into  contact.  If  through 
the  intracellular  nature  of  the  poisons  and  the  mildly  invasive 
character  of  the  micro-organisms  this  action  is  restricted 
to  the  seat  of  original  infection,  a  local  manifestation  will 
result.  Its  exact  nature  will,  however,  be  modified  to  some 
extent  by  other  qualities  of  the  bacterial  products.  Thus, 
when  in  addition  to  their  irritative  action  which,  when  mild, 
occasions  multiplication  of  the  cells  of  the  connective  and 
lymphoid  tissues,  and,  when  extreme,  effects  the  death  of 
the  cells,  the  products  are  strongly  chemotactic,  suppuration 
will  occur. 

Fever  and  suppuration  are,  therefore,  non-specific  actions, 
because  numerous  micro-organisms  share  the  qualities  pro- 
ductive of  these  conditions  in  common. 

If  the  bacteria  are  rapidly  invasive,  but  still  have  injurious 
products  of  the  intracellular  variety,  they  are  apt  to  share 
certain  qualities,  such  as  the  swelling  of  the  lymph-nodes, 
etc.,  in  common,  so  that  such  lesions  cannot  be  considered  as 
specific.  So  soon  as  any  one  of  the  products  is  discovered  to 
give  some  single  lesion  peculiar  to  that  organism  by  which 
it  is  produced,  or  so  soon  as  the  total  effect  of  the  activity 
of  the  various  products  of  any  micro-organism  produces  a 


Specific  Affinity  of  the  Cells  for  the  Toxins      93 

typical  effect,  differing  from  the  total  effect  of  the  operation 
of  other  micro-organisms,  so  that  a  recognized  type  of  dis- 
ease results,  it  becomes  possible  to  say  that  the  micro-organ- 
ism in  question  is  specific. 

The  most  striking  examples  of  the  specific  action  of 
bacterio-toxins  is,  however,  seen  in  those  cases  where  soluble 
extracellular  metabolic  products  of  bacterial  energy  are 
liberated  into  the  body  juices  so  as  to  be  conveyed  by  the 
circulatory  system  to  all  parts  of  the  body.  Those  cells 
most  susceptible  to  its  action  are  then  first  or  most  pro- 
foundly impressed  by  it,  and  definite  responses  brought 
about.  Thus,  the  soluble  toxin  of  tetanus  causes  no  visible 
reaction  in  the  cells  with  which  it  first  comes  into  contact 
at  the  seat  of  primary  infection,  because  these  cells  are 
either  less  susceptible  to  its  influence,  or  are  less  well  able 
to  show  its  effects,  than  the  cells  of  the  nervous  system  to 
which  it  is  secondarily  carried  by  the  blood. 

SPECIFIC  AFFINITY  OF  THE  CELLS  FOR  THE  TOXINS. 

The  cells  of  the  connective  tissue  in  which  the  tetanus 
bacillus  is  living  show  little  reaction,  but  the  motor  cells  of 
the  central  nervous  system,  having  a  greater  affinity  for  it, 
are  profoundly  impressed,  so  that  convulsions  of  the  con- 
trolled muscular  system  are  brought  about.  This  special 
excitation  of  the  nerve  cells  is  specific  because  no  other 
bacterio-toxin  is  known  to  produce  it  and  it  is  attributed 
to  special  selective  affinities  of  the  nerve  cells  for  the  poison. 
This  affinity  has  its  analogue  among  the  poisons  of  higher 
plants,  thus,  strychnin  has  a  similar  selective  affinity  and  is 
also  said  to  be  specific  in  action  upon  the  motor  cells. 

The  venoms  of  various  serpents,  especially  the  cobra,  also 
have  specific  reactions,  the  cells  of  the  respiratory  centers 
seeming  to  be  most  profoundly  affected  by  them. 

The  diphtheria  bacillus,  when  observed  in  ordinary  throat 
infections,  is  seen  to  produce  a  pseudomembranous  angina 
which  results  in  part  from  an  irritative  local  action  of  the 
organism,  which  it  shares  in  common  with  many  others, 
and  in  part  from  some  coagulating  product  which  it  shares 
in  common  with  a  few — pneumococcus,  streptocococus,  etc. 
Neither  of  these  reactions  is  specific,  but  subsequent  to  these 
early  manifestations  comes  depressant  action  on  the  nervous 
cells  with  palsy,  peculiar  to  the  products  of  the  diphtheria 
bacillus,  and  therefore  specific. 


94  Infection 

It  is  upon  the  peculiar  specific  reactions  of  the  bacterio- 
toxins  and  the  peculiar  susceptibility  of  certain  cells  to  this 
action  that  the  production  of  distinct  clinical  manifestations 
depend. 

THE  INVASION  OF  THE  BODY  BY  MICRO-ORGANISMS. 

Some  bacteria  whose  invasiveness  is  insufficient  to  enable 
them  successfully  to  maintain  life  in  healthy  tissues,  occa- 
sionally get  a  foothold  in  diseased  tissues  and  assist  in  morbid 
changes.  This  is  sometimes  seen  in  what  is  described  by 
the  surgeons  as  sapremia,  in  which  various  saprophytic 
bacteria,  possessing  no  invasive  powers,  by  growing  in  the 
putrefying  tissues  of  a  gangrenous  part,  give  rise  to  poison- 
ous substances  which  when  absorbed  by  the  adjacent 
healthy  tissues  produce  constitutional  disturbances,  such 
as  depression,  fever,  and  the  like. 

Bacteria  with  limited  invasive  powers  and  intracellular 
toxins  can  at  best  occasion  local  inflammatory  effects. 
Such  organisms  not  infrequently  vary,  however,  and  when 
of  unusual  vitality  may  survive  entrance  into  the  blood  and 
lymph  circulations  and  occasion  bacteremia,  or,  as  it  is  more 
frequently  called,  septicemia,  a  morbid  condition  charac- 
terized by  the  presence  of  bacteria  in  the  circulating  blood. 
When  bacteria  entering  into  the  circulation  are  unable  to 
pervade  the  entire  organisms  and  continue  in  the  circulation, 
they  may  collect  in  the  capillaries  of  the  less  resisting  tissues, 
producing  local  metastatic  lesions,  usually  purulent  in 
character.  This  form  of  invasion  results  in  what  is  surgically 
known  as  pyemia. 

The  mode  by  which  the  entrance  of  bacteria  into  the 
circulation  is  effected  differs  in  different  cases.  Kruse  * 
believes  that  they  sometimes  are  passively  forced  through 
the  stomata  of  the  vessels  when  the  pressure  of  the  inflam- 
matory exudate  is  greater  than  that  of  the  blood  within 
them;  that  they  may  sometimes  enter  in  the  bodies  of 
leukocytes  that  have  incorporated  them;  that  they  may 
actually  grow  through  the  capillary  walls,  or  that  they  reach 
the  blood  circulation  indirectly  by  first  following  the  course 
of  the  lymphatics. 

Toxemia  results  from  the  absorption  of  the  poisonous 
bacterial  products  from  non-invasive  bacteria,  as  in  tetanus. 

*  Fliigge,  "Die  Mikroorganismen, "  vol.  i,  p.  271. 


The  Cardinal  Conditions  of  Infection          95 


THE  CARDINAL  CONDITIONS  OF  INFECTION. 

Infection  can  take  place  only  when  the  micro-organisms 
are  sufficiently  virulent,  when  they  enter  in  sufficient  num- 
ber, when  they  enter  by  appropriate  avenues,  and  when  the 
host  is  susceptible  to  their  action. 

Virulence. — Virulence  may  be  defined  as  the  disease- 
producing  power  of  micro-organisms.  It  is  a  variable 
quality,  and  depends  upon  the  invasiveness  of  the  bacteria, 
or  the  toxicity  of  their  products,  or  both. 

A  few  bacteria  are  almost  constant  in  virulence  and  can 
be  kept  under  artificial  conditions  for  years  with  very  little 
change.  Other  bacteria  begin  to  diminish  in  virulence  so 
soon  as  they  are  introduced  to  the  artificial  conditions  of 
life  in  the  test-tube.  Still  others,  and  perhaps  the  greater 
number,  can  be  modified,  and  their  virulence  increased  or 
diminished  according  to  the  experimental  manipulations 
to  which  they  are  subjected. 

Variation  in  virulence  is  not  always  a  peculiarity  of  the 
species,  for  the  greatest  differences  may  be  observed  among 
individuals  of  the  same  kind.  Thus,  the  streptococcus 
usually  attenuates  rapidly  when  kept  in  artificial  media, 
so  that  special  precautions  have  to  be  taken  to  maintain  it, 
but  Hoist  observed  a  culture  whose  virulence  was  unaltered 
after  eight  years'  continuous  cultivation  in  the  laboratory 
without  any  particular  attention  having  been  devoted  to  it. 
What  is  true  of  different  cultures  of  the  same  organisms,  is 
equally  true  of  the  individuals  in  the  same  culture.  To 
determine  such  individual  differences  is  quite  easy  among 
chromogenic  bacteria.  If  these  are  plated  in  the  ordinary 
way  it  will  be  found  that  some  colonies  are  paler  and  some 
darker  than  others.  Conn  found  that  by  repeating  the 
plating  a  number  of  times  and  always  selecting  the  palest 
and  darkest  colonies  he  was  eventually  able  to  produce  two 
cultures,  one  brilliant  yellow,  the  other  colorless,  from  the 
same  original  stock. 

Decrease  of  virulence  under  artificial  conditions  prob- 
ably depends  upon  artificial  selection  of  the  organisms  in 
transplantation  from  culture  to  culture.  When  planted 
upon  artificial  media,  the  vegetative  members  of  the  bacte- 
rial family  proceed  to  grow  actively  and  soon  exceed  in 
number  their  more  pathogenic  fellows.  Each  time  the 
culture  is  transplanted,  more  of  the  vegetative  and  fewer 


96  Infection 

of  the  pathogenic  forms  are  carried  over,  until  after  the 
organism  is  accustomed  to  its  new  environment,  and  grows 
readily  upon  the  artificial  media,  it  is  found  that  the  patho- 
genic organisms  have  been  largely  or  entirely  eliminated  and 
the  vegetative  forms  alone  retained. 

Increase  of  virulence  can  be  achieved  by  artificial 
selection  so  planned  as  to  preserve  the  more  virulent  or 
pathogenic  organisms  at  the  same  time  that  the  less  virulent 
and  more  vegetative  organisms  are  eliminated.  In  cases 
in  which  no  virulence  remains,  the  experimental  manipula- 
tion of  the  culture  is  directed  toward  gradual  immunization 
of  the  micro-organisms  to  the  defensive  mechanisms  of  the 
body  of  the  animal  for  which  the  organism  is  to  be  made 
virulent.  A  number  of  methods  are  made  use  of  for  this 
purpose. 

Passage  Through  Animals. — Except  in  cases  where  the 
virulence  of  the  micro-organism  is  invariable,  it  is  usually 
observed  that  the  transplantation  of  the  organism  from 
animal  to  animal  without  intermediate  culture  in  vitro 
greatly  augments  its  pathogenic  power.  Of  course,  this 
artificially  selects  those  members  of  the  bacterial  family  best 
qualified  for  development  in  the  animal  body,  eliminating 
the  others,  and  the  virulence  correspondingly  increases. 

The  increase  in  virulence  thus  brought  about  is,  however, 
not  so  much  an  increase  in  the  general  pathogenic  power  of 
the  organism  for  all  animals,  as  toward  the  particular  animal 
or  kind  of  animal  used  in  the  experiments.  Thus,  in  general, 
the  passage  of  bacteria  through  mice  increases  their  virulence 
for  mice,  but  not  necessarily  for  cats  or  horses;  passage 
through  rabbits,  the  virulence  for  rabbits,  but  not  necessarily 
for  dogs  or  pigeons,  etc. 

This  specific  character  of  the  virulence  is  well  accounted 
for  by  the  "  lateral-chain  theory  of  immunity,"  where  it  will 
again  be  considered. 

The  Use  of  Collodion  Sacs. — When  cultures  of  bacteria 
are  enclosed  in  collodion  sacs  and  placed  in  the  abdominal 
or  other  body  cavities  of  animals,  and  kept  in  this  manner 
through  successive  generations,  the  virulence  is  usually  con- 
siderably increased.  This  is  one  of  the  favorite  methods 
used  by  the  French  investigators.  It  keeps  the  bacteria  in 
constant  contact  with  the  slightly  modified  body  juices  of 
the  animal,  which  transfuse  through  the  collodion,  and  thus 
impedes  the  development  of  such  organisms  as  are  not  able 


The  Cardinal  Conditions  of  Infection          97 

to  endure  their  injurious  influences.  Thus  it  becomes  only 
another  way  of  carrying  on  an  artificial  selection  of  those 
members  of  the  bacterial  family  that' can  endure,  and  elimi- 
nating those  that  cannot  endure  the  defensive  agencies  of 
those  juices  with  which  the  organisms  come  in  contact.* 

The  addition  of  animal  fluids  to  the  culture-media  some- 
times enables  the  investigator  to  increase,  and  usually 
enables  him  to  maintain,  the  virulence  of  bacteria.  The 
cultivation  of  the  organism  should  embrace  a  series  of  genera- 
tions in  gradually  increasing  concentrations  of  the  body 
fluid  employed,  until  the  organism  becomes  thoroughly 
accustomed  to  it. 

In  some  cases  it  may  be  sufficient  to  use  a  single  standard 
mixture,  thus:  Shaw  f  found  that  he  could  exalt  the  viru- 
lence of  anthrax  bacilli  by  cultivating  them  upon  blood- 
serum  agar  for  fourteen  generations,  after  which  they  were 
three  times  as  active  as  cultures  similarly  transferred  upon 
ordinary  agar-agar. 

The  increase  under  such  conditions  as  these  probably 
depends  upon  the  immunization  of  the  bacteria  to  the  body 
juices  of  the  animals,  and  this  whole  matter  will  be  under- 
stood after  the  subject  "Immunity"  has  been  considered. 

Number. — The  number  of  bacteria  entering  the  infected 
animal  has  a  very  important  bearing  upon  infection,  and 
may  itself  determine  whether  it  shall  occur  or  not. 

The  entrance  of  a  single  micro-organism  of  any  kind  is 
scarcely  ever  able  to  effect  infection  because  of  the  uncer- 
tainty of  its  being  able  to  withstand  the  defensive  mechanisms 
of  the  animal  into  which  it  is  introduced.  In  most  cases 
a  considerable  number  of  organisms  is  necessary  in  order 
that  some  may  survive  the  new  environment.  Park  points 
out  that  when  bacteria  are  transplanted  from  culture  to 
culture,  under  conditions  supposed  to  be  favorable,  many 
of  them  die.  It  seems  not  improbable,  therefore,  that  when 
they  are  transplanted  to  an  environment  in  which  are  present 
certain  mechanisms  for  defending  the  organism  against  them, 
many  more  must  inevitably  die.  The  more  virulent  an 
organism  is,  the  fewer  will  be  the  number  required  to  infect. 
Marmorek,  in  his  experiments  with  antistreptococcic  serum, 
used  a  streptococcus  whose  virulence  was  exalted  by  passage 

*  Directions  for  making  and  using  the  capsules  are  given  in  the 
chapter  upon  Animal  Experimentation, 
f  "Brit.  Med.  Jour.,"  May  9,  1903. 

7 


98  Infection 

through  rabbits  and  intermediate  cultivation  upon  agar- 
agar  containing  ascitic  fluid,  until  one  hundred  thousand 
millionth  of  a  cubic  centimeter  (un  cent  milliardieme)  was 
fatal  for  a  rabbit.  In  this  quantity  it  is  scarcely  probable 
that  more  than  a  single  coccus  could  have  been  present. 
Single  anthrax  or  glanders  bacilli  may  infect  rabbits  and 
guinea-pigs.  Roger  found  that  820  tubercle  bacilli  from  the 
culture  with  which  he  experimented  were  required  to  infect 
a  guinea-pig,  when  introduced  beneath  the  skin.  Herman 
found  that  it  required  4  or  5  c.c.  of  a  culture  of  Staphylococ- 
cus  pyogenes  to  produce  suppuration  in  the  peritoneal  cavity 
of  an  animal;  0.75  c.c.  to  produce  it  beneath  the  skin;  0.25 
c.c.  in  the  pleura;  0.05  c.c.  in  the  veins  and  o.oooi  c.c.  in  the 
anterior  chamber  of  the  eye. 

In  experimenting  with  Bacillus  proteus  vulgaris,  Watson 
Cheyne  found  that  5,000,000  to  6,000,000  organisms  injected 
beneath  the  skin  did  not  produce  any  lesion ;  8,000,000  caused 
the  formation  of  an  abscess;  56,000,000  produced  a  phleg- 
mon from  which  the  animal  died  in  five  or  six  weeks  and 
225,000,000  were  required  to  cause  the  death  of  the  animal 
in  twenty-four  hours.  In  studying  Staphylococcus  aureus 
upon  rabbits  he  found  that  25,000,000  would  cause  an 
abscess,  but  1,000,000,000  were  necessary  to  cause  death. 

The  Avenue  of  Infection. — The  successful  invasion  of 
the  body  by  certain  bacteria  can  be  achieved  only  when 
they  enter  it  through  appropriate  avenues.  Even  when 
invasion  is  possible  through  several  channels,  the  parasite 
most  commonly  invades  through  one  that  may,  therefore, 
be  regarded  as  most  appropriate,  invasion  through  which 
furnishes  the  typical  picture  of  the  infection. 

Thus,  gonococci  usually  reach  the  body  through  the  uro- 
genital  mucous  membranes,  where  they  set  up  the  various 
inflammatory  reactions  collectively  known  as  gonorrhea — 
i.  e.,  urethritis,  vaginitis,  prostatitis,  orchitis,  cystitis,  etc. 
These  constitute  the  typical  picture  of  infection.  The 
organism  may  also  successfully  invade  the  conjunctiva, 
producing  blennorrhea,  but  there  is  no  evidence  that  gono- 
cocci can  successfully  invade  the  body  through  the  skin, 
the  respiratory,  or  alimentary  mucous  membrane. 

Typhoid  and  cholera  infections  seem  to  take  place  through 
the  alimentary  mucous  membrane,  and  the  evidence  that 
infection  takes  place  by  inhalation  is  slight.  It  is  not  known 


The  Cardinal  Conditions  of  Infection          99 

to  take  place  through  the  urogenital  system,  the  conjunctiva, ' 
or  the  skin. 

The  avenue  of  entrance  not  only  determines  infection, 
but  may  also  determine  the  form  that  it  takes.  Thus, 
tubercle  bacilli  rubbed  into  the  deeper  layer  of  the  skin 
produce  a  chronic  inflammatory  disease,  called  lupus, 
that  lasts  for  years  and  rarely  results  in  generalized  tubercu- 
losis. Bacilli  reaching  the  cervical  or  other  lymph-nodes  by 
entrance  through  the  tonsils ,may  remain  localized,  produc- 
ing enlargement  and  softening  of  the  nodes,  or  passing 
through  them  reach  the  circulation,  in  which  they  may  be 
carried  to  the  bones  and  joints  and  occasion  chronic  inflam- 
mation with  necrosis  and  ultimate  evacuation  or  exfolia- 
tion of  the  diseased  mass,  after  which  the  patient  may 
recover.  Bacilli  entering  the  intestine  in  many  cases  pro- 
duce implantation  lesions  in  the  intestinal  walls;  bacilli 
inhaled  into  the  lung,  or  conveyed  to  it  from  the  intestine 
by  the  thoracic  duct  and  veins,  produce  the  ordinary  pul- 
monary tuberculosis  known  as  phthisis  or  consumption. 

Inhaled  pneumococci  colonizing  in  the  pharynx  have  been 
known  to  produce  pseudomembranous  angina;  in  the  lungs, 
pneumonia ;  implanted  upon  the  conjunctiva,  conjunctivitis. 
Jn  these  cases  we  can  look  upon  the  type  of  infection  as 
depending  upon  the  portal  through  which  the  invading 
organism  found  its  way  into  the  tissues. 

The  avenue  of  entrance  is,  for  obvious  reasons,  less 
important  when  the  micro-organism  is  of  the  rapidly  invasive 
form,  whose  chief  operation  is  in  the  streaming  blood  or  in 
the  lymphatics.  Anthrax  in  most  animals  is  characterized 
by  a  bacteremia  regardless  of  the  point  of  primary  infection. 
Bubonic  plague  rapidly  becomes  a  bacteremia  regardless  of 
the  entrance  of  the  Bacillus  pestis  by  inhalation  into  the 
lungs,  or  by  way  of  the  lymphatics  through  superficial 
lesions.  The  failure  of  the  micro-organisms  to  colonize 
successfully  when  introduced  through  inappropriate  avenues 
may  be  explained  by  a  consideration  of  the  local  conditions 
to  which  they  are  subjected. 

When  they  are  introduced  beneath  the  skin,  bacteria  are, 
in  most  cases,  delayed  in  reaching  the  circulation,  and  are 
in  the  meantime  subjected  to  the  germicidal  action  of  the 
lymph  and  exposed  to  the  attacks  of  phagocytes.  Many 
succumb  to  these  and  never  penetrate  more  deeply  into  the 
body.  Should  any  survive,  they  may  be  transported  to 


100 


Infection 


'the  lymph-nodes  and  there  destroyed,  or,  passing  through 
these  barriers  without  destruction,  and  reaching  the  venous 
channels,  they  have  next  to  pass  through  the  pulmonary  cap- 
illaries, where  they  are  apt  to  be  caught  and  destroyed.  Fi- 
nally, should  any  escape  all  these  defenses  and  reach  the 
general  circulation,  it  is  to  find  the  endothelium  of  the 
capillaries  prone  to  collect  and  detain  them  until  destruction 
is  finally  effected.  The  systemic  circulation  is  also  defended 
against  such  micro-organisms^  as  might  reach  the  veins 
through  lesions  or  accidents  of  the  abdominal  viscera,  by 
the  interposition  of  the  portal  capillary  network  of  the  liver, 
where  the  bacteria  are  caught  and  many  of  them  destroyed, 
or  passing  which,  the  pulmonary  capillary  system  acts  as  a 
second  barrier  against  them.  The  deeper  the  penetration,  the 
more  active  the  defense  becomes,  the  blood  itself  furnishing 
agglutinins,  bacterio-lysins,  and  phagocytes  for  the  destruc- 
tion of  the  micro-organisms  and  the  protection  of  the  host. 

These  defenses,  however,  are  of  no  avail  against  actively 
invasive  organisms  provided  with  the  means  of  overcoming 
them  all  through  aggressins  that  destroy  the  germicidal 
humors  or  toxins  that  kill  or  paralyze  the  cells.  When 
these  are  injected  directly  into  the  streaming  blood  they 
produce  their  effects  more  rapidly  than  when  injected 
beneath  the  skin  or  elsewhere,  because  the  field  of  operation 
is  immediately  reached  instead  of  through  a  roundabout 
course  in  which  so  many  defenses  have  to  be  overcome. 
Taking  anthrax  bacilli,  whose  invasiveness  has  already  been 
dwelt  upon,  as  an  example,  Roger  *  found  that  when  the 
organisms  were  injected  into  the  aorta,  animals  died  more 
quickly  than  when  they  were  injected  into  the  veins  and 
obliged  to  find  their  way  through  the  pulmonary  capillaries 
to  the  general  circulation.  If  the  injections  were  made  into 
the  portal  vein,  the  animals  stood  a  good  chance  of  recovery, 
the  liver  possessing  the  power  of  destroying  sixty-four 
times  as  many  anthrax  bacilli  as  would  prove  fatal  if  intro- 
duced through  other  channels. 

The  conditions  differ,  however,  in  different  infections, 
for  when  Roger  experimented  with  streptococci  instead  of 
anthrax  bacilli,  he  found  that  if  the  bacilli  were  inoculated 
into  the  portal  vein  the  animals  died  more  quickly  than 
when  they  were  injected  into  the  aorta,  and  that  when 
the  bacilli  were  injected  into  the  peripheral  veins  the  ani- 
*" Introduction  to  the  Study  of  Medicine,"  p.  151. 


The  Cardinal  Conditions  of  Infection        101 

mals  lived  longest,  the  liver  seeming  to  be  far  less  destruc- 
tive to  streptococci  than  the  lungs. 

The  Susceptibility  of  the  Host.— Susceptibility  is  lia- 
bility to  infection.  It  is  a  conditon  in  which  the  host  is 
unable  to  defend  itself  against  invading  micro-organisms. 
Unusual  or  unnatural  susceptibility  is  also  spoken  of  as 
predisposition  or  dyscrasia. 

Many  animals  and  plants  are  naturally  without  any  means 
of  overcoming  the  invasiveness  of  certain  parasitic  micro- 
organisms, and  are,  therefore,  naturally  susceptible;  others 
naturally  resist  their  inroads,  but  through  various  temporary 
or  permanent  physiologic  changes  may  lose  the  defensive 
power. 

In  general,  it  is  true  that  any  condition  that  depresses  or 
diminishes  the  general  physiological  activity  of  an  animal 
diminishes  its  ability  to  defend  itself  against  the  pathogenic 
action  of  bacteria,  and  so  predisposes  to  infection.  These 
changes  are  often  so  subtile  that  they  escape  detection, 
though  at  times  they  can  be  partly  understood. 

The  inhalation  of  noxious  -vapors.  It  has  long  been 
supposed  that  sewer  gas  was  responsible  for  the  occurrence 
of  certain  infectious  diseases,  and  when  the  nature  of  these 
diseases  was  made  clear  by  a  knowledge  of  their  bacterial 
causes,  the  old  belief  still  remained  and  many  sanitarians 
continued  to  believe  that  defective  sewage  is  in  some  way 
connected  with  their  occurrence.  It  is  difficult  to  prove 
or  disprove  the  matter  experimentally.  Men  who  work 
in  sewers  and  plumbers  who  breathe  much  sewer  gas  are 
not  apparently  affected  by  it.  Alessi*  found  that  rats, 
rabbits,  and  guinea-pigs  kept  in  cages  some  of  which  were 
placed  over  the  opening  of  a  privy,  while  in  others  the 
excreta  of  the  animals  were  allowed  to  accumulate,  suffered 
from  a  pronounced  diminution  of  the  resisting  powers. 
This  would  seem  to  be  inconsistent  with  the  habits  of  rats, 
many  of  which  live  in  sewers.  Abbott  f  caused  rabbits  to 
breathe  air  forced  through  sewage  and  putrid  meat  infusions 
for  one  hundred  and  twenty-nine  days,  and  found  that  the 
products  of  decomposition  inhaled  by  the  animals  played  no 
part  in  producing  disease,  or  in  inducing  susceptibility  to  it. 

Fatigue  is  a  well-recognized  clinical  cause  of  suscep- 
tibility to  disease,  and  experimental  evidence  of  its 

*"Centralbl.  f.  Bakt.,"  etc.,  1894,  xv,  p.  228. 
t"  Trans.  Assoc.  Amer.  Phys.,"  1895. 


102  Infection 

correctness  is  not  wanting.  Charrin  and  Roger*  found  that 
white  rats,  which  naturally  resist  infection  with  anthrax, 
succumbed  to  the  infection  if  compelled  to  turn  a  revolving 
wheel  until  exhausted  before  inoculation. 

Exposure  to  cold  seriously  influences  the  resisting  power 
of  the  warm-blooded  animals.  It  is  an  everyday  expe- 
rience that  chilling  the  body  predisposes  to  "  cold "  and 
may  be  the  starting-point  of  pneumonia.  Pasteur  found 
that  fowls,  which  resist  anthrax  under  normal  conditions, 
succumbed  to  infection  if  kept  for  some  time  in  a  cold  bath 
before  inoculation. 

The  reverse  seems  to  be  true  of  the  cold-blooded  animals, 
for  Gibierf  found  that  frogs,  naturally  resistant  to  the 
anthrax  bacillus,  would  succumb  to  infection  if  kept  at  37°  C. 
after  inoculation. 

Diet  produces  some  variation  in  the  resisting  powers. 
The  tendency  of  scorbutics  to  suffer  from  infectious  dis- 
orders of  the  mouth,  the  frequency  with  which  epidemics 
of  infectious  disease  follow  famines,  and  the  enterocolitis  of 
marasmatic  infants,  illustrate  the  effects  of  insufficient  food 
in  predisposing  to  disease.  We  also  find  that  the  infectious 
diseases  of  carnivorous  animals  are  not  the  same  as  those  of 
herbivorous  animals,  and  that  the  former  are  exempt  from 
many  disorders  to  which  the  latter  quickly  succumb. 
Hankin  was  able  to  show  experimentally  that  meat-fed  rats 
resisted  anthrax  infection  far  better  than  rats  fed  upon 
bread. 

Intoxication  of  all  kinds  predisposes  to  infection.  Plata- 
niaj  found  that  such  animals  as  frogs,  pigeons,  and  dogs 
became  susceptible  to  anthrax  when  under  the  influence 
of  curare,  chloral,  and  alcohol.  Leo§  found  that  white  rats 
fed  upon  phloridzin  became  susceptible  to  anthrax.  Wag- 
ner ||  found  that  pigeons  become  susceptible  to  anthrax  when 
under  the  influence  of  chloral.  Abbott**  found  the  resisting 
powers  of  rabbits  against  Streptococcus  pyogenes  and 

*  "  Compte  rendu  Soc.  de  Biol  de  Paris,"  Jan.  24,  1890. 

f'Compte  rendu  Acad.  des  Sciences  de  Paris,"  1882,  t.  xcix,  p. 
1605. 

JSee  Sternberg's  "  Immunity  and  Serum  Therapy,"  p.  10;  "  Cen- 
tralbl.  f.  Bakt.,"  etc.,  Bd.  vn,  p.  405. 

§  "  Zeitschrift  fiir  Hyg.,"  Bd.  vn,  p.  505,  1889. 

||  "  Wratsch,"  1890,  39,  40. 
**  "  Jour,  of  Exp.  Med.,"  vol.  i,  No.  3,  1896. 


Mixed  Infections  103 

Bacillus  coli  diminished  by  daily  intoxication  with  5  to 
15  c.c.  of  alcohol  introduced  into  the  stomach  through  a  tube. 
Salant*  found  that  alcohol  was  disadvantageous  in  com- 
batting the  infectious  diseases  because  it  diminished  the 
glycogen  content  of  the  liver  which  Collaf  had  found  an 
important  adjunct  in  supporting  the  resisting  power. 

It  is  a  common  clinical  observation  that  excessive  indul- 
gence in  alcohol  predisposes  to  certain  infections,  notably 
pneumonia,  and  every  surgeon  knows  the  danger  of  pneu- 
monia after  anesthetization  with  ether. 

Traumatic  injury  and  mutilation  of  the  body  are  not 
without  effect  upon  infection.  The  more  extensive  the 
damage  done  to  the  tissues,  the  greater  the  danger  of  infec- 
tion, and  the  more  serious  the  consequences  of  infection  when 
it  takes  place. 

The  mutilation  of  the  body  by  the  removal  of  certain 
organs  is  of  disputed  importance.  There  is  much  literature 
upon  the  effect  of  the  spleen  in  overcoming  infectious  agents, 
but  the  experimental  evidence  seems  about  equally  divided 
as  to  whether  an  animal  is  more  or  less  susceptible  after 
the  removal  of  this  organ  than  it  was  before. 

Morbid  conditions  in  general  predispose  to  infection. 
The  frequency  with  which  diabetics  suffer  from  furuncles, 
carbuncles,  and  local  gangrenous  lesions  of  the  skin;  the 
increased  susceptibility  of  phthisics  to  bronchopneumonia 
of  other  than  tuberculous  origin ;  the  apparent  predisposition 
of  injured  joints  and  pneumonic  lungs  to  tuberculosis;  the 
extensive  streptococcus  invasions  accompanying  scarlatina 
and  variola ;  the  presence  of  Bacillus  icteroides  and  various 
other  organisms  in  the  blood  and  tissues  of  yellow  fever 
patients,  and  the  presence  of  Bacillus  sui  pestifer  in  the 
bodies  of  hogs  suffering  with  hog  cholera,  all  show  the 
diminution  in  the  general  resisting  power  of  an  individual 
already  diseased. 

MIXED  INFECTIONS. 

The  general  prevalence  of  bacteria  determines  that  few 
can  enter  and  infect  the  body  of  a  host  without  the  associa- 
tion of  other  kinds.  Therefore  their  operation  in  the  body 
is  subject  to  modifications  produced  in  them  or  in  the  host 
by  these  associated  organisms. 

*  "Jour.  Amer.  Med.  Assoc.,"  XLVII,  18,  Nov.  3,  1906,  p.  1467. 
t  "  Archiv.  Ital.  de  Biologic,"  xxvi. 


104  Infection 

In  experimental  investigations  this  fact  is  not  infrequently 
forgotten,  and  it  is  often  remarked  with  surprise  that  the 
results  of  inoculation  with  pure  cultures  of  a  micro-organism 
may  be  clinically  different  from  those  observed  under  natural 
conditions. 

The  tetanus  bacillus,  which  endures  with  difficulty  the 
effects  of  uncombined  oxygen,  flourishes  in  association  with 
saprophytic  organisms  by  which  the  oxygen  is  absorbed. 
The  same  thing  is  probably  true  of  other  obligatory  anaerobic 
organisms. 

The  metabolic  products  of  one  species  may  intensify  or 
accelerate  the  action  of  those  of  an  associated  species,  or 
the  reverse  may  be  true,  and  the  products  of  different  organ- 
isms, having  different  chemical  composition,  may  neutralize 
one  another,  or  combine  to  form  some  entirely  new  sub- 
stance which  is  entirely  different  from  its  antecedents. 
Such  conditions  cannot  fail  to  influence  the  type  and  course 
of  infection. 


CHAPTER  IV. 
IMMUNITY. 

IMMUNITY  is  ability  to  resist  infection.  It  is  the  ability 
of  an  organism  successfully  to  antagonize  the  invasive 
powers  of  parasites,  or  to  annul  the  injurious  properties  of 
their  products.  The  mechanism  of  immunity  is  compli- 
cated or  otherwise  according  to  circumstances.  When  the 
invasive  action  of  non-toxicogenic  bacteria  is  to  be  over- 
come, certain  reactions,  mostly  on  the  part  of  the  phagocytic 
cells,  are  called  into  action;  when  the  toxic  products  of 
bacteria  are  to  be  deprived  of  injurious  effects,  the  reaction 
seems  to  take  place  between  the  toxin  and  certain  com- 
bining and  neutralizing  substances  contained  in  the  body 
juices;  when  bacterial  invasion  and  intoxication  are  both 
to  be"  antagonized,  both  mechanisms  are  engaged  in  the 
defenses,  comparatively  simple  or  exceedingly  complex, 
according  to  the  conditions  involved.  The  more  involved  the 
conditions  of  infection  become,  the  more  complicated  the 
defensive  reactions  become,  until  it  may  no  longer  be  pos- 
sible accurately  to  analyze  them. 

Some  have  endeavored  to  refer  all  of  the  phenomena  of 
immunity  to  the  ability  of  the  animal  to  endure  the  bacterio- 
toxins,  and  have  sought  to  relegate  the  reactions  against 
invasion  to  a  subsidiary  place.  This  is  undoubtedly  an 
error,  as  the  mechanisms  are  different  and  the  prompt  action 
of  one  may  make  the  action  of  the  other  unnecessary. 
Metschnikoff*  found  that  frogs  injected  with  0.5  c.c.  of 
cholera  toxin  died  promptly,  but  that  frogs  injected  with 
cultures  of  the  cholera  spirillum  recovered  without  illness. 
This  would  suggest  that  the  recovery  of  the  infected  frog 
depended  upon  some  defensive  mechanism  combating  the 
invasiveness  of  the  bacteria  and  so  preventing  the  produc- 
tion of  the  toxin  to  which  the  frog  was  susceptible. 

Immunity  must  not  be  conceived  as  something  insepar- 

*  "  Immunite  dans  les  Maladies  Infectieuses,"  Paris,  1901,  p.  150. 

105 


io6  Immunity 

ably  associated  with  infection.  The  reactions  of  the  body 
toward  bacteria  in  the  infectious  diseases  are  identical  with 
those  toward  other  minute  irritative  bodies,  and  the  reactions 
toward  bacterio-toxins  are  identical  with  those  toward  other 
active  substances,  so  that  the  only  way  by  which  a  sat- 
isfactory understanding  of  the  phenomena  can  be  reached 
is  by  carefully  comparing  the  reactions  produced  by  bacteria 
and  their  products  with  those  produced  by  other  active 
bodies. 

Immunity  is  called  active  when  the  animal  protects  itself 
through  its  own  activities,  passive  when  its  protection 
depends  upon  defensive  substances  prepared  by  some  other 
animal  and  forced  upon  it.  Thus,  if  a  frog  be  injected  with 
anthrax  bacilli,  the  leukocytes  devour  the  bacteria,  destroy 
them,  and  so  protect  the  frog  from  infection,  the  immunity 
is  active  because  it  depends  upon  the  activity  of  the  frog's 
phagocytes.  But  if  a  guinea-pig  previously  given  anti- 
tetanic  serum  be  injected  with  tetanus  toxin,  and  so  recovers 
from  the  toxin,  the  resisting  power,  conferred  by  the  anti- 
toxin previously  injected,  does  not  depend  upon  any  activity 
of  the  animal,  which  remains  entirely  passive. 

Immunity  is  largely  relative.  Fowls  are  immune  against 
tetanus,  that  is,  they  can  endure,  without  injury,  as  much 
toxin  as  tetanus  bacilli  can  produce  in  their  bodies,  and 
suffer  no  ill  effects  from  inoculation.  If,  however,  a  large 
quantity  of  tetanotoxin  produced  in  a  test-tube  be  intro- 
duced into  their  bodies,  they  succumb  to  it.  Mongooses 
and  hedgehogs  are  sufficiently  immune  against  the  venoms 
of  serpents  to  resist  as  much  poison  as  is  ordinarily  injected 
by  the  serpents,  but  by  collecting  the  venom  from  several 
serpents  and  injecting  considerable  quantities  of  it,  both 
animals  can  be  killed.  Rats  cannot  be  killed  by  injection 
with  Bacillus  diphtheriae,  and  Cobbett*  found  that  they 
could  endure  from  1500  to  1800  times  as  much  diphtheria 
toxin  as  guinea-pigs,  though  more  than  this  would  kill  them. 

Carl  Fraenkel  has  expressed  the  whole  matter  very 
forcibly  when  he  says:  "A  white  rat  is  immune  against 
anthrax  in  doses  sufficiently  large  to  kill  a  rabbit,  but  not 
necessarily  against  a  dose  sufficiently  large  to  kill  an  ele- 
phant." 

*  "Brit.  Med.  Jour.,"  April  15,  1899. 


Natural  Immunity  107 


NATURAL  IMMUNITY. 

Natural  immunity  is  the  natural,  inherited  resistance 
against  infection  or  intoxication,  peculiar  to  certain  groups 
of  animals,  and  common  to  all  the  individuals  of  those 
groups. 

Few  micro-organisms  are  capable  of  affecting  all  varieties 
of  animals;  indeed,  it  is  doubtful  whether  any  known 
organism  possesses  such  universally  invasive  powers. 

The  micro-organisms  of  suppuration  seem  able  to  infect 
animals  of  many  different  kinds,  sometimes  producing  local 
lesions,  sometimes  invading  rapidly  with  resulting  bactere- 
mia.  The  tubercle  bacillus  is  known  to  be  pathogenic  for 
mammals,  birds,  reptiles,  batrachians,  and  fishes,  though  it 
is  still  uncertain  whether  the  infecting  organisms  in  these 
cases  are  identical  or  slightly  differing  species. 

As  a  rule,  however,  the  infectivity  of  bacteria  and  other 
micro-organisms  is  restricted  to  certain  groups  of  animals 
which  usually  have  more  or  less  resemblance  to  one  another ; 
thus,  anthrax  is  essentially  a  disease  of  warm-blooded  ani- 
mals, though  certain  exceptions  are  observed,  and  Metschni- 
koff  has  found  that  hippocampi  (sea-horses),  perch,  crickets, 
and  certain  mussels  are  susceptible.  Among  the  warm- 
blooded animals  anthrax  is  most  frequent  among  the  her- 
bivora,  though  some  carnivora  may  also  be  infected. 

Close  relationship  is  not,  however,  a  guarantee  that 
animals  will  behave  similarly  toward  infection.  The  rabbit, 
guinea-pig,  and  the  rat  are  rodents,  but  though  the  rabbit 
and  guinea-pig  are  susceptible  to  anthrax,  the  rat  is  immune. 
This  is  still  better  exemplified  in  the  susceptibility  of  mice 
to  glanders.  The  field-mouse  seems  to  be  the  most  suscep- 
tible of  all  animals  to  infection  with  Bacillus  mallei;  the 
house  mouse  is  much  less  susceptible,  and  the  white  mouse 
is  immune.  Mosquitos,  though  closely  related,  are  differ- 
ent in  their  immunity  to  the  malarial  parasite.  The  culex 
does  not  harbor  the  parasite  at  all,  and  of  the  anopheles, 
two  very  similar  species  seem  to  behave  very  differently, 
Anopheles  maculipennis  being  the  common  definitive  host 
of  the  parasite,  while  Anopheles  punctipennis  is  not  known 
to  be  susceptible  to  it.  The  same  differences  may  exist  among 
the  members  of  the  human  species.  It  has  been  asserted 
that  Mongolians,  and  especially  Japanese,  are  immune 


io8  Immunity 

against  scarlatina,  and  that  negroes  are  immune  against 
yellow  fever,  but  increasing  information  is  to  the  contraryc 

Human  beings  suffer  from  typhoid,  cholera,  measles, 
scarlatina,  yellow  fever,  varicella,  and  numerous  other 
diseases  unknown  among  the  lower  animals,  even  those 
domestic  animals  with  which  they  come  in  close  contact. 
They  also  suffer  from  Malta  fever,  anthrax,  rabies,  glanders, 
bubonic  plague,  and  tuberculosis,  which  are  common  among 
the  lower  animals.  Animals,  in  turn,  suffer  from  distemper, 
hog  cholera,  Texas  fever,  swine-plague,  chicken  cholera, 
mouse  septicemia,  etc.,  the  respective  micro-organisms  of 
which  are  not  known  to  infect  man. 

It  has  already  been  pointed  out  that  mongooses  and 
hedgehogs  are  immune  against  the  venom  of  serpents  from 
which  other  animals  quickly  die.  The  tobacco-worm  lives 
solely  upon  tobacco-leaves,  the  juice  of  which  is  intensely 
poisonous  to  higher  animals,  and  is  also  a  good  insecticide. 
Boxed  cigars  and  baled  tobacco  are  often  ruined  by  the 
larvae  of  a  small  beetle  that  feeds  upon  them,  and  a  glance 
over  the  poisonous  vegetables  will  show  that  few  of  them 
escape  the  attacks  of  insects  immune  against  their  juices. 

These  facts  are  sufficient  to  show  that  many  animals 
are  by  nature  immune  against  the  invasion  of  microparasites 
of  certain  kinds,  and  that  they  are  also  at  times  immune 
against  poisons.  Immunity  against  one  kind  of  infection 
or  intoxication  is,  however,  entirely  independent  of  all  other 
infections  and  intoxications.  Immunity  against  infection 
usually  guarantees  exemption  from  the  toxic  products  of 
that  particular  micro-organism,  though  experiment  may 
show  the  animal  to  be  susceptible  to  it.  Immunity  against 
any  form  of  bacterio-toxin  usually,  though  not  necessarily, 
determines  that  the  micro-organism,  though  it  may  be  able 
to  invade  the  body,  can  do  very  little  harm. 

ACQUIRED  IMMUNITY* 

Acquired  immunity  is  resistance  against  infection  or  in- 
toxication possessed  by  certain  animals,  of  a  naturally  sus- 
ceptible kind,  in  consequence  of  conditions  peculiar  to  them 
as  individuals.  It  is  a  peculiarity  of  the  individual,  not  of 
his  kind,  and  signifies  a  subtile  change  in  physiology  by 
which  latent  defensive  powers  are  stimulated  to  action. 
The  reactions  in  general  correspond  with  those  of  natural 
immunity,  and  comprise  mechanisms  for  overcoming  the 


Acquired  Immunity  109 

invasion  of  pathogenic  organisms,  for  neutralizing  or  destroy- 
ing their  toxins  or  both.  As  an  acquired  character  and  an 
individual  peculiarity  it  is  not  transmitted  to  the  offspring, 
though  these  sometimes  also  acquire  immunity  through  the 
parents.  Thus  in  studying  immunity  of  mice  against  ricin, 
Ehrlich  found  that  the  newly  born  offspring  of  an  immune 
mother  were  not  immune,  though  they  subsequently  became 
so  through  the  mother's  milk. 

Acquired  immunity  differs  from  natural  immunity  in 
being  less  certain  and  of  variable  duration.  The  animal 
may  be  immune  to-day,  but  lose  all  power  of  defending 
itself  a  month  hence. 

Natural  immunity  is  always  active,  but  certain  forms 
of  acquired  immunity  are  passive. 

Immunity  may  be  acquired  through  infection  or  intoxica- 
tion, and  either  of  these  may  be  accidental  or  experimental. 

(A)  Active  Acquired  Immunity. — i.  Immunity  Ac- 
quired through  Infection. — (a)  Accidental  Infection.— 
The  most  familiar  form  of  acquired  immunity  follows  an 
attack  of  an  infectious  disease.  Every  one  knows  that  an 
attack  of  measles,  scarlatina,  varicella,  variola,  yellow  fever, 
typhoid  fever,  and  other  common  infectious  maladies,  is  a 
fairly  good  guarantee  of  future  exemption  from  the  respec- 
tive disease.  Immunity  thus  acquired  is  not  transmissible 
to  the  offspring.  Almost  everybody  has  had  measles,  yet 
almost  all  children  are  born  susceptible  to  it.  It  is  not 
necessarily  permanent,  as  is  shown  by  the  not  infrequent 
cases  in  which  second  attacks  of  measles  occur.  In  some 
cases,  as  after  typhoid  fever,  the  immunity  is  not  at  first  ob- 
servable and  the  patient  may  suffer  from  relapses.  Later  it 
becomes  well-established  and  no  repetition  of  the  disease  is 
possible  for  years. 

Sometimes  the  infection,  by  which  immunity  is  acquired, 
is  not  exactly  similar  to  the  disease  against  which  it  affords 
protection,  as  in  the  case  of  vaccinia,  which  protects  against 
variola.  It  is  still  controversial,  however,  whether  cow-pox 
is  variola  of  the  cow  or  an  entirely  different  disease.  Cow- 
pox  was,  however,  common  in  the  days  when  smallpox 
was  frequent,  and  has  now  become  an  extremely  rare 
disease. 

(b)  Experimental  Infection. — i.  Inoculation:  The  oldest 
experiments  in  immunity  date  from  unknown  antiquity  and 
were  practised  in  China  and  other  Oriental  countries  for  the 


no  Immunity 

purpose  of  preventing  smallpox.  The  Chinese  method  of 
experimentally  producing  variolous  infection  was  very  crude 
and  consisted  in  introducing  crusts  from  cases  of  variola 
into  the  nose,  and  tying  them  upon  the  skin.  The  Turkish 
method  was  much  more  neat,  in  that  a  small  quantity  of 
the  variolous  pus  was 'introduced  into  a  scarification  upon 
the  skin  of  the  individual  to  be  protected.  Lady  Montague, 
wife  of  the  British  Ambassador,  brought  the  so-called 
"inoculation"  method  of  preventing  smallpox  from  Turkey 
in  the  early  part  of  the  eightheenth  century  (1718).  By  both 
methods  the  very  disease,  variola,  against  which  protection 
was  desired,  was  occasioned,  the  only  advantage  of  the 
experimental  over  the  accidental  infection  being  that  by 
selecting  the  infective  virus  from  a  mild  case  of  variola,  by 
performing  the  operation  at  a  time  when  no  epidemic  of  the 
disease  was  raging,  and  by  doing  it  at  a  time  when  the  person 
infected  was  in  the  most  perfect  physical  condition,  the  dan- 
gers of  the  malady  might  be  mitigated. 

There  was  always  danger,  however,  that  the  induced 
disease  being  true  variola  might  prove  unexpectedly  severe, 
and  that  each  inoculated  individual,  suffering  from  the  con- 
tagious disease,  might  start  an  epidemic. 

2.  Jennenan  vaccination:  In  1791  a  country  schoolmaster 
named  Plett,  living  in  the  town  of  Starkendorf  near  Kiel  in 
Germany,  seems  to  have  made  the  first  endeavor  to  subject 
the  oft-repeated  observation  that  persons  who  had  acquired 
cow-pox  did  not  subsequently  become  infected  with  small- 
pox to  experimental  demonstration  by  inserting  cow-pox 
virus  into  three  children,  all  of  whom  escaped  smallpox. 

The  father  of  vaccination,  and  the  man  to  whom  the  world 
owes  one  of  its  greatest  debts,  was  Edward  Jenner,  who 
performed  his  first  experiment  on  May  14,  1796,  when  he 
transferred  some  of  the  contents  of  a  cow-pox  pustule  on 
the  arm  of  a  milkmaid  named  Sarah  Nelmess  to  the  arm  of 
a  boy  named  John  Phips.  After  the  lad  had  recovered  from 
the  experimental  cow-pox  thus  produced,  he  subsequently 
introduced  smallpox  pus  into  his  arm  and  found  him  fully 
immunized  and  rendered  insusceptible  to  the  disease.  This 
led  Jenner  to  perform  many  other  experiments,  and  record 
his  observations  in  numerous  scientific  memoirs.  The 
success  of  his  work  immediately  attracted  the  attention  of 
both  scientific  investigators  and  sanitarians,  and  its  out- 
come has  been  the  establishment  of  compulsory  vaccination 


Vaccination  1 1 1 

by  legal  enactment  in  nearly  all  civilized  countries,  with 
the  result  that  smallpox,  instead  of  being  one  of  the  most 
prevalent  and  most  dreaded  diseases,  has  become  one  of  the 
most  rare  and  least  feared. 

The  immunity  acquired  through  vaccination  is  active  and 
usually  of  prolonged  duration.  It  is  subject  to  the  same 
variations  observed  in  other  experimentally  acquired  im- 
munities, these  variations  explaining  the  occasional  failures 
which  constitute  the  "stock  in  trade"  of  those  who  still 
remain  unconvinced  of  the  scientific  basis  and  efficacy  of 
the  procedure. 

Though  a  thorough  analysis  of  the  irregularities  and  ex- 
ceptions of  vaccination  would  be  of  much  interest,  a  brief 
mention  of  the  most  important  must  suffice  for  the  present 
argument. 

The  first  controversial  point  is  the  nature  of  the  "vaccine," 
or  virus  used  in  the  operation.  It  is  obtained  from  calves 
or  heifers  suffering  from  experimental  cow-pox,  and  is  a  virus 
descended  from  various  spontaneous  cases  of  cow-pox 
observed  in  places  remote  from  one  another.  Experts  are 
undecided  whether  cow-pox  is  variola*  modified  by  passage 
through  the  cow  so  that  the  transplanted  micro-organisms 
are  only  capable  of  inducing  a  local  instead  of  a  general 
disease,  or  whether  it  is  an  independent  affection  natural  to 
the  cow. 

In  reality  the  matter  in  unimportant,  so  long  as  the  desired 
effect  is  accomplished,  and  the  true  lineage  of  the  virus  is 
only  a  matter  of  scientific  curiosity.  As  immunity  is  almost 
invariably  a  specific  effect  resulting  from  infection,  it  would 
seem  most  likely  that  cow-pox  and  smallpox  were  originally 
identical. 

The  advantage  of  "vaccination"  over  "inoculation"  is 
that  the  induced  disease  is  local  and  not  dangerous  except  in 
rare  cases,  and  that  it  is  not  contagious.  The  natural  varia- 
tions in  the  susceptibility  of  different  vaccinated  individuals 
determine  that  a  few  persons  cannot  be  successfully  vacci- 
nated, being  immune  to  the  mildly  invasive  organisms  of 
vaccinia,  though  perhaps  susceptible  to  the  actively  in- 
vasive organisms  of  variola;  that  a  few  individuals  shall 
prove  abnormally  susceptible  to  vaccinia  so  that  the  disease 
departs  from  its  usual  local  type  and  generalizes,  but  that 
in  nearly  all  cases  the  disease  will  follow  the  well-known  type 
of  a  local  lesion  characterized  by  definite  periods  of  incu- 
bation, vesiculation,  pustulation,  and  cicatrization. 


ii2  Immunity 

The  occasional  variations  in  immunity  of  different  in- 
dividuals also  determine  that  having  been  vaccinated  once 
an  individual  may  not  again  become  susceptible  to  vaccina- 
tion, though  he  may  become  susceptible  to  the  more  actively 
invasive  organisms  of  variola,  or  that  he  may  soon  become 
again  susceptible  to  both  diseases,  or  that  in  very  rare  cases 
no  immunity  against  variola  will  result  from  vaccination. 
In  most  cases  successful  vaccination  can  be  repeated  once 
or  twice  at  intervals  of  seven  or  ten  years,  and  experience 
shows  that  the  immunity  against  smallpox  conferred  by 
vaccination  is  of  longer  duration  and  usually  becomes  per- 
manent after  vaccination  has  been  repeated  once  or  twice. 

Sanitarians  are  accustomed  to  speak  of  efficient  and 
inefficient  vaccination.  These  are  vague  terms  and  do  not 
seem  to  be  understood  by  the  laity.  Efficient  vaccination 
is  vaccination  repeated  as  often  as  is  necessary.  It  has 
already  been  shown  that  individual  variations  determine 
that  a  few  individuals  never  become  immune,  hence  never 
can  be  efficiently  vaccinated.  Other  persons  are  efficiently 
vaccinated  by  a  single  operation.  The  term  is  usually  inter- 
preted to  indicate  that  which  experience  has  shown  to  be 
efficient  in  average  cases. 

Failures  not  uncommonly  result  from  causes  having 
nothing  to  do  with  the  problems  of  immunity.  That  an 
operation  of  scarification  has  been  performed  upon  a  child, 
and  that  a  scar  has  remained  thereafter  may  mean  nothing. 
It  is  not  the  operation  but  the  disease  that  achieves  the 
result,  and  if  the  operation  be  improperly  done,  poor — i.  e., 
old  or  inert — matter  introduced,  or  if  after  introduction  it 
be  destroyed  by  the  application  of  antiseptics,  no  effect  can 
be  expected.  Hence  all  persons  that  have  been  vaccinated 
may  not  have  had  vaccinia,  the  essential  condition  leading 
to  immunity.  Nor  does  the  occurrence  of  a  local  lesion  act 
as  a  guarantee  that  vaccinia  has  been  induced.  Careful 
examination  of  the  resulting  lesions  should  always  be  made, 
that  the  type  of  the  infection  may  be  studied.  It  is  the 
disease,  vaccinia,  that  must  occur — three  days  incubation, 
three  days  vesiculation,  three  days  pustulation,  and  subse- 
quent cicatrization  with  the  formation  of  a  punctate  scar. 

An  arm  may  be  never  so  sore,  may  suppurate  or  even 
become  gangrenous,  without  vaccinia  having  occurred  or 
the  desired  benefit  attained. 

The  accidents  of  vaccination  were  formerly  numerous  and 


Vaccination  113 

sometimes  disastrous  because  of  the  general  inattention  to 
the  quality  of  the  materials  used,  the  mode  of  inserting  them, 
the  condition  of  the  patient's  skin,  and  the  careless  treat- 
ment of  the  resulting  lesions.  When  human  virus  was  used, 
the  transmission  of  human  diseases,  such  as  syphilis  and 
erysipelas,  occasionally  took  place ;  now  these  are  rare  acci- 
dents indeed.  When  no  attention  was  paid  to  the  quality  of 
the  bovine  virus,  and  no  governmental  inspection  of  labor- 
atories required,  the  accidental  contamination  of  the  virus 
occasioned  a  small  number  of  accidental  infections  of  the 
patients'  arms,  but  these  evils  are  becoming  less  and  less  as 
greater  attention  is  given  to  the  details  of  the  process. 
Some  accidents  and  some  few  deaths  there  will  probably 
always  be,  just  as  there  are  occasional  accidents  and  occa- 
sional fatal  results  following  all  kinds  of  trivial  injuries, 
though  care  will  eliminate  them  as  the  sources  of  accident 
are  better  understood. 

3.  Pasteurian  vaccination  or  bacterination:  Although  the 
word  vaccination  is  derived  from  the  Latin  vacca,  "  a  cow," 
and  was  first  employed  in  connection  with  Jenner's  method  of 
introducing  virus  modified  by  passage  through  a  cow,  Pasteur, 
in  honor  of  Jenner,  applied  it  to  every  kind  of  protective 
inoculation,  and  the  word  bacterination  is  only  introduced  for 
the  purpose  of  indicating  certain  differences  in  the  method. 

In  1880  Pasteur*  observed  that  some  hens  inoculated 
with  a  culture  of  the  bacillus  of  chicken  cholera  that  had 
been  on  hand  for  some  time  did  not  die  as  was  expected. 
Later,  securing  a  fresh  and  virulent  culture,  these  and 
other  chickens  were  inoculated.  The  former  hens  did 
not  die,  the  new  hens  did.  Quick  to  observe  and  study 
phenomena  of  this  kind,  he  investigated  and  found  that 
when  chickens  we're  inoculated  with  old  and  non-virulent 
cultures  they  acquired  immunity  against  virulent  cultures. 
This  led  him  to  the  recommendation  of  the  employment  of 
attenuated  cultures  as  vaccines  against  the  disease,  and  to  the 
achievement  of  great  success  in  preventing  epidemics  by 
which  great  numbers  of  the  barnyard  fowls  of  France  were 
being  destroyed. 

In  1 88 1  Pasteur,!  m  experimenting  with  Bacillus  anthracis, 
observed  that  if  the  organism  were  cultivated  at  unusually 
high  temperatures  it  lost  the  power  of  producing  spores,  and 

*  "Compte  rendu  de  la  Soc.  de  Biol.,  1880,  239;  315  et  seq. 

t  "  Compte  rendu  de  la  Soc.  de  Biol.  de  Paris,"  1881,  xcn,  pp.  662-665. 


ii4  Immunity 

diminished  in  virulence.  He  also  found  that  when  the  organ- 
isms had  been  so  attenuated,  they  could  not  regain  virulence 
without  artificial  manipulation.  It  occurred  to  him  that 
such  organisms,  possessing  feeble  virulence,  might  be  able 
to  confer  immunity  upon  animals  into  which  they  were 
inoculated,  and  he  continued  to  investigate  the  subject  until 
he  found  that  by  using  three  "  vaccines"  or  modified  cultures 
of  increasing  virulence,  it  was  possible  to  render  animals 
immune  against  the  unmodified  organisms.  This  method 
was  put  to  practical  test  with  great  success,  and  has  since 
been  extensively  practised  in  different  parts  of  the  world. 

Arloing,  Cornevin  and  Thomas,*  and  Kittf  found  that 
exposure  of  the  Bacillus  anthracis  symptomatici  to  a  high 
temperature  in  the  dry  state  modified  its  virulence  and 
devised  a  practical  method  of  protecting  cattle  against 
symptomatic  anthrax  by  inoculating  them  with  powdered 
muscle  tissue  containing  the  bacilli  attenuated  by  drying 
and  exposure  to  85  °  C.  This  method  has  since  been  in  use 
in  many  countries,  and  has  given  excellent  satisfaction. 

In  1889  Pasteur,  {  continuing  his  researches  upon  the 
experimental  modification  of  the  germs  of  disease  and  their 
use  as  prophylactics,  published  his  famous  work  upon  rabies, 
and  showed  that,  although  the  micro-organism  of  that 
disease  had  so  far  eluded  discovery,  it  was  contained  in  the 
central  -nervous  system  of  diseased  animals,  where  it  could 
be  modified  in  virulence  by  drying.  By  placing  spinal  cords 
removed  from  rabid  rabbits  in  a  glass  jar  containing  cal- 
cium chlorid,  he  was  able  to  diminish  the  virulence  of  the 
contained  micro-organisms  according  to  the  duration  of  the 
exposure.  The  introduction  of  the  attenuated  virus  was  fol- 
lowed by  the  development  of  a  certain  degree  of  immunity. 
By  repeated  inoculation  of  more  and  more  active  viruses 
animals  acquired  complete  immunity  against  street  virus. 
These  experiments  formed  the  basis  of  the  "  Pasteur  method" 
of  treating  rabies,  which  is  nothing  more  than  immunization 
with  the  modified  germs  of  the  disease  during  the  long  incuba- 
tion period  of  the  disease. 

Haffkine§  found  that  the  introduction  of  killed  cultures 
of  virulent  cholera  spirilla  produced  immunity  against  the 
living  micro-organisms,  and  used  the  method  with  consider- 

"Le  Charbon  Symptomatique  du  Boeuf,"  Paris,  1887. 
t  "Centralbl.  f.  Bakt.,"  etc.,  i,  p.  684. 

t  "Compte  rendu  de  la  Soc.  de  Biol.  de  Paris,"  1881,  cvm,  p.  1228. 
§"Brit.  Med.  Jour.,"  1891,  n,  p.  1278. 


Immunity  Acquired  by  Intoxication          115 

able  success  for  preventing  the  disease.  Later*  he  applied 
the  same  method,  also  with  considerable  success,  for  the  pre- 
vention of  bubonic  plague,  and  A.  E.  Wright  f  followed  pretty 
much  the  same  method  for  the  prevention  of  typhoid  fever. 

In  all  these  cases  the  immunity  induced  by  the'experimen- 
tal  manipulations  is  specific  in  nature,  and  variable  in  inten- 
sity, according  to  the  method  of  treatment  adopted  and  the 
thoroughness  with  which  it  is  carried  out.  This  variability 
in  the  results  attained  will  be  much  better  understood  after 
the  subject  of  immunization  against  toxins  has  been  discussed. 

2.  Immunity  Acquired  by  Intoxication. — Bacterio-toxins 
form  a  miscellaneous  group  of  active  bodies  of  entirely 
different  chemical  composition  and  physiologic  activity. 
Some  are  toxalbumins,  some  are  enzymes,  some  are  bac- 
terio-proteins.  The  true  nature  of  the  greater  number 
of  these  bodies  is  unknown,  but  study  of  their  physio- 
logic action  has  brought  forth  the  important  fact  that 
their  behavior  toward  the  body  cells  is  in  no  way  different 
from  the  behavior  of  the  same  cells  toward  other  chemical 
compounds  of  similar  constitution,  and  that  nearly  all 
physiologically  active  bodies  introduced  into  living  organisms 
produce  definite,  though  not  necessarily  visible,  reactions. 

Such  reactions  are  now  known  as  antigenic,  and  the  sub- 
stances by  which  they  are  induced  have  been  called  by 
Deutsch  antigens. I  Since  its  introduction  the  precise 
meaning  given  the  word  by  Deutsch  has  been  slightly 
changed.  As  now  defined,  an  antigen  is  any  protein  sub- 
stance which  when  injected  into  the  body  of  a  living  organism 
is  capable  of  producing  a  chemicophysiologic  reaction  re- 
sulting in  the  appearance  of  a  self-neutralizing,  self-precipi- 
tating, self-agglutinating,  self-dissolving,  or  otherwise  self- 
antagonizing  substance  known  as  an  antibody. 

The  antigens  are,  so  far  as  known,  all  colloidal  substances. 
They  may  be  harmful  or  harmless,  active  or  inert,  living  or 
dead,  organized  or  unorganized.  The  reactions  are  specific 
and  the  antibody  has  specific  affinity  for  that  antigen  alone 
by  which  its  formation  has  been  excited. 

All  poisonous  substances  are  not  antigens,  even  though  a 
certain  immunity — in  the  sense  of  habituation  or  tolerance — 
may  follow  their  repeated  administration.  One  may  become 

*  "Brit.  Med.  Jour.,"  1895,  n,  p.  1541. 
t  Ibid.,  Jan.  30,  1897,  i,  p.  256. 

J  Deutsch  and  Feistmantel,  "Die  Impfstoffe  und  Sera,"  1903,  Leip- 
zig, Thieme. 


n6  Immunity 

habituated  or  tolerant  to  a  certain  quantity  of  mercury  or  ar- 
senic, and  to  certain  alkaloids,  such  as  morphin,  caffein,  nico- 
tin,  cocain,  etc.,  but  he  does  not  react  as  to  antigens  and  no 
antibodies  are  formed.  To  these  various  substances  he 
acquires  only  a  slight  degree  of  tolerance;  to  the  injurious 
effects  of  antigens  he  may  acquire  an  almost  unlimited  degree 
of  immunity  through  the  formation  of  the  antibodies. 

From  remote  antiquity  it  has  been  known  that  those  who 
regularly  consumed  small  quantities  of  poisons  become  ir- 
responsive to  their  action,  and  it  is  well  known  that  Mithrid- 
ates  adopted  this  mode  of  defending  himself  from  his  enemies. 

Chauveau*  believed  that  the  immunity  conferred  by  in- 
oculations of  bacteria  was  due  to  the  presence  of  their  soluble 
products,  but  the  first  direct  demonstration  was  given  by 
Salmon  and  Smith,!  who,  as  early  as  1886,  showed  that  it 
was  possible  to  immunize  pigeons  against  the  hog-cholera 
bacillus  by  means  of  repeated  injection  with  cultures  exposed 
to  60°  C.,  and  containing  no  living  organisms.  CharrinJ 
found  it  possible  to  immunize  rabbits  against  Bacillus  pyo- 
cyaneus  by  injecting  them  with  the  filtered  products  of  cul- 
tures of  that  organism,  and  Bonome§  similarly  to  immunize 
animals  against  Bacillus  proteus,  B.  cholera  gallinarum  and 
the  pneumococcus.  Roux  and  Chamberland||  and  Roux** 
were  able  by  the  use  of  boiled  cultures  of  the  bacilli  of  malig- 
nant edema,  and  of  quarter  evil,  similarly  to  immunize 
animals  against  these  respective  infections. 

The  subject  was  much  further  elaborated  by  Roux  and 
Yersinff  in  their  experiments  with  diphtheria  toxin,  by 
Behringit  in  his  early  studies  of  diphtheria,  and  by  Kita- 
sato§§  in  his  experiments  with  tetanus. 

These  early  experiments  opened  a  wide  field,  through  the 
investigation  of  which  we  now  know  that  the  products  as  well 
as  the  living  or  dead  bacteria  of  most  of  the  infectious  diseases, 
when  properly  introduced  into  animals,  can  induce  immunity. 

(B)  Passive  Acquired  Immunity. — Passive  immunity  is 
always  acquired,  never  natural.  It  depends  upon  defensive 

*"Ann.  de  1'Inst.  Pasteur,"  1888,  2. 

t  "Centralbl.  f.  Bakt.,"  etc.,  1887,  n,  No.  18,  p.  543. 

t"Compte  rendu,"  cv,  p.  756. 

§  "Zeitschrift  f.  Hyg.,"  v,  p.  415. 

||  "Ann.  de  1'Inst.  Pasteur,"  1887,  12. 
**  Ibid.,  1888,  2.  ft  Ibid.,  n,  1888,  p.  629. 

It  "Deutsche  med.  Wochenschrift,"  1890,  No.  50. 
§§  "Zeitschrift  fur  Hygiene,"  x,  1891,  p.  267. 


Passive  Acquired  Immunity  117 

factors  not  originating  in  the  animal  protected,  but  arti- 
ficially or  experimentally  supplied  to  it.  The  fundamental 
principle  is  simple  and  has  become  the  basis  of  serum  thera- 
peutics. If  the  immunized  animal  generates  factors  by 
which  the  infecting  bacteria  can  be  destroyed  or  the  activity 
of  their  products  overcome  in  its  body,  cannot  these  fac- 
tors be  removed  and  the  benefit  they  confer  transferred  to 
another  animal? 

The  first  experiments  in  this  direction  seem  to  have 
been  made  by  Babes  and  Lepp,*  who  found  that  the 
blood-serum  of  animals  immunized  to  rabies  showed  a 
defensive  power  when  injected  into  other  animals.  Ogata 
and  Jasuharaf  found  that  the  subcutaneous  injection  of 
blood-serum  from  an  animal  immunized  against  anthrax 
enabled  the  injected  animals  successfully  to  resist  infection. 
Behring  and  KitasatoJ  found  that  the  blood-serums  of 
animals  immunized  against  diphtheria  and  tetanus,  when 
mixed  with  cultures  of  these  respective  bacilli,  neutralized 
their  power  to  produce  disease.  Kitasato  §  found  that  if 
mice  were  inoculated  with  tetanus  bacilli,  they  could  be 
saved  from  the  fatal  infection  by  the  infra-abdominal  in- 
jection of  some  blood-serum  from  a  mouse  immunized 
against  tetanus,  even  after  symptoms  of  the  disease  had 
appeared.  Ehrlich||  showed  that  the  blood-serums  of  animals 
immunized  against  abrin  and  ricin  could  save  other  animals 
from  the  fatal  effects  of  these  respective  toxalbumins ;  Phis- 
alix  and  Bertrand,**  and,  later,  Calmetteft  found  the  blood- 
serum  of  animals,  immunized  against  the  venoms  of  serpents, 
similarly  possessed  the  power  of  neutralizing  the  poisonous 
effects  of  the  venoms.  KosselJJ  found  that  the  blood-serum 
of  animals,  immunized  against  the  poisonous  blood-serum  of 
eels,  contained  a  body  which  destroyed  or  neutralized  the 
effects  of  the  eels'  serum. 

Thus,  it  is  shown  that  in  each  case  in  which  defensive  reac- 
tions are  stimulated  in  experiment  animals,  these  reactions  are 
accompanied  by  the  appearance  in  the  blood-serum  of  those 

*  "Annales  de  1'Inst.  Pasteur,"  1889,  vol.  m. 

t  "Centralbl.  f.  Bakt.,"  etc.,  ix,  p.  25,  1890. 

|  "Deutsche  med.  Woch.,"  1890,  No.  49. 

§  "Zeitschrift  fur  Hygiene,"  1892,  xu,  p.  256. 

)|  "Deutsche  med.  Wochenschrift,"  1891,  Nos.  32  and  44. 
**  "Compte  rendu  Acad.  des  Sciences  de  Paris,"  cxvm,  p.  556. 
ft  "Ann.  de  1'Inst.  Pasteur,"  1894,  vm,  p.  275. 
}J  "Berliner  klin.  Woch.,"  1898,  p.  152. 


n8  Immunity 

animals  of  factors  that  can  be  utilized  to  defend  other  ani- 
mals in  whose  bodies  no  similar  reactions  have  been  produced. 

Passive  immunity  may  also  be  brought  about  in  a  few 
cases  by  the  injection  into  the  intoxicated  animal  of  sub- 
stances, other  than  immunity  products,  that  have  a  specific 
affinity  for  the  poison.  Thus  Wassermann  and  Takaki* 
found  that  when  the  crushed  spinal  cord  of  a  rabbit  was 
mixed  in  -vitro  with  tetanus  toxin,  the  poison  was  quickly 
absorbed  by  the  nerve-cells,  so  that  the  mixture  became 
inert  and  could  be  injected  into  animals  without  harm. 
Wassermann  also  found  that  the  same  effects  could  be  pro- 
duced in  the  bodies  of  animals,  and  that  when  the  crushed 
spinal  cord  was  injected  into  an  animal  twenty-four  hours, 
or  a  few  hours  previously,  or  a  few  hours  after  a  fatal  dose  of 
tetanus  toxin,  enough  of  the  combining  elements  remained 
in  the  blood  to  fix  the  toxin  before  it  anchored  itself  to  the 
central  nervous  system  of  the  intoxicated  animal.  Myersf 
found  that  the  ground-up  tissue  of  the  adrenal  bodies  was 
able  to  fix  and  thus  annul  the  poisonous  effects  of  cobra 
venom  in  vitro. 

In  all  these  cases  the  neutralizing  effects  are  either  accom- 
plished or  initiated  by  factors  prepared  experimentally,  and 
forced  upon  the  animal  in  whose  body  their  activities  are 
manifested. 


SYNOPSIS   OF  THE    EXPERIMENTAL   STUDIES    OF 
IMMUNITY. 

Very  important  contributions  were  made  by  Ehrlich,t  in 
his  work  upon  the  vegetable  toxalbumins,  ricin,  abrin,  and 
robin.  Kossel§  investigated  the  reactions  produced  with 
toxic  eels'  blood  and  found  that  immunity  could  be  estab- 
lished against  their  hemolytic  action.  Phisalix  and  Ber- 
trand||  showed  that  immunity  could  also  be  produced  in 
guinea-pigs  against  the  action  of  viper  venom. 

The  investigations  upon  other  active  bodies  were  soon 
begun.  In  1893  Hildebrand  **  studied  emulsin  and  found 
that  it  produced  a  definite  reaction  with  the  formation,  in 

*  "Berliner  klin.  Wochenschrift,"  Jan.  3,  1898. 
t  "Lancet,"  July  2,  1898. 

t  "Deutsche  med.  Woch.,"  1891,  Nos.  32  and  44. 
§  "Berliner  klin  Wochenschrift,"  1898. 

j|  Atti  d  XI  Congr.  med.  internaz.  Roma,  1894,  u,  200-202. 
**  "Virchow's  Archives,"  Bd.  cxxxi. 


Synopsis  of  Experimental  Studies  of  Immunity    119 

animals  injected,  of  an  anti-emulsin.  v.  Dungern  *  studied 
proteolytic  enzymes  of  various  bacteria,  and  showed  that 
when  gelatin-dissolving  enzymes  were  repeatedly  injected 
into  animals,  definite  reactions  took  place,  and  in  the  serum 
a  body  appeared  that  inhibited  the  action  of  the  ferment  in 
a  test-tube.  Gheorghiewskif  immunized  animals  to  cul- 
tures of  Bacillus  pyocyaneus,  and  found  that  the  reaction 
provoked  caused  the  appearance  in  the  serum  of  some  body 
that  prevented  the  formation  of  the  blue  pigment  so  char- 
acteristic of  the  organism.  MorgenrothJ  applied  the  same 
principle  to  rennet,  finding  that  it  produced  definite  reac- 
tions, with  the  formation  of  an  anti-body  inhibiting  the 
coagulation  of  milk.  Bordet  and  Gengoul  found  that  the 
fibrin  ferment  of  the  blood  of  one  animal  was  active  in  the 
body  of  another  animal,  producing  an  inhibiting  substance 
by  which  the  coagulation  of  the  blood  of  the  first  animal 
could  be  delayed. 

The  studies  of  Krausll  showed  a  new  fact,  that  when 
filtered  cultures  of  the  cholera  spirillum  were  introduced 
into  animals,  the  serum  of  these  animals,  added  to  the  filtered 
culture  in  a  test-tube,  caused  the  appearance  of  a  delicate 
flocculent  precipitate. 

Wassermann  and  Schiitze**  found  that  when  cows'  milk 
was  repeatedly  injected  into  rabbits,  their  serum  acquired 
the  property  of  occasioning  a  precipitate  when  added  to 
cows'  milk,  but  not  when  added  to  goats'  or  any  other 
milk.  If,  however,  the  rabbit  had  been  repeatedly  injected 
with  goats'  milk  or  human  milk,  its  serum  would  precipitate 
with  those  milks  respectively,  and  not  with  cows'  milk. 
The  reaction  was  thus  shown  to  be  specific. 

Meyers  ft  found  that  the  repeated  intraperitoneal  injec- 
tion of  egg-albumen  into  rabbits  caused  their  serum  to  give 
a  dense  precipitate  when  added  to  solutions  of  egg-albumen. 

Tchistowitch  **  found  that  eels'  serum  injected  into  ani- 
mals produced  a  reaction  in  which  immunity  to  its  poison- 
ous action  was  associated  with  the  ability  of  their  serum  to 
produce  a  precipitate  when  added  to  the  eels'  serum. 

*  "Miinchener  med.  Woch.,"  Aug.  15,  1898. 

t  "Ann.  de  1'Inst.  Pasteur,"  1899. 

t  "Centralbl.  f.  Bakt.,"  etc.,  1899,  xxvi,  p.  349. 

§  "Ann.  de  1'Inst.  Pasteur,"  1903,  xvn,  p.  822. 

||  "Wien.  klin.  Woch.,"  1897. 
**  "Deutsche  med.  Woch.,"  1900. 
ft  "Lancet,"  n,  1900.         JJ  "Ann.  de  1'Inst.  Pasteur,"  vol.  13. 


120  Immunity 

Closely  connected  with  these  various  reactions  are  certain 
others  variously  spoken  of  as  cytotoxic,  cytolytic,  hemolytic, 
bacteriolytic,  etc.  The  first  observation  bearing  upon  these 
was  made  by  R.  Pfeiffer,*  who  found  that  when  guinea-pigs 
received  frequent  intraperitoneal  injections  of  cholera  spirilla 
and  became  thoroughly  immunized,  their  serum  behaved 
very  peculiarly  toward  the  bacteria  in  the  peritoneal  cavity 
of  freshly  infected  animals,  in  that  it  caused  them  to  become 
aggregated  into  granular  masses  and  subsequently  to  dis- 
appear. This  became  known  as  "  Pfeiffer 's  phenomenon." 
The  serum  of  the  immunized  animal  was  devoid  of  action 
by  itself,  the  serum  of  the  infected  animal  was  inactive,  but 
the  combination  of  the  two  brought  about  dissolution  of 
the  micro-organisms.  Later  it  was  shown  by  Metschnikofff 
that  the  living  animal  was  not  a  factor  in  the  process,  but 
that  what  was  seen  in  the  peritoneal  cavity  could  be  re- 
produced in  a  test-tube,  though  not  quite  as  well. 

BordetJ  made  frequent  injections  of  defibrinated  rabbits' 
blood  into  guinea-pigs,  and  obtained  a  serum  that  had  a 
solvent  action  upon  the  rabbit's  corpuscles  in  -vitro,  and 
showed  that  the  induced  hemolysis  resembled  in  all  points 
the  bacteriolysis. 

Ehrlich  and  Morgenroth§  studied  the  hemolytic  action 
of  the  serum  of  goats  that  had  been  frequently  injected  with 
the  defibrinated  blood  of  sheep  and  goats,  and  were  able  to 
point  out  the  mechanism  of  the  corpuscle  solution  or  hemo- 
lysis. It  was  found  to  depend  upon  two  associated  factors, 
one  of  which,  the  lysin  or  solvent,  was  present  in  normal 
blood,  and  was  called  "addiment"  or  "complement,"  and 
another  present  only  in  the  serum  of  the  reactive  animals, 
called  the  "immune  body"  or  "intermediate  body." 
The  former  was  labile  and  easily  destroyed  by  heat,  the 
latter  stabile  and  not  affected  by  heat  up  to  the  point  of 
coagulation.  The  experiments  were  confirmed  by  von 
Diingern  and  many  others.  It  is  to  be  observed  in  passing 
that  this  reaction  differs  from  the  direct  solution  of  the  cor- 
puscles in  vitro  by  cobralysin,  which  was  studied  by  Myers,  1 1 
and  tetanolysin,  studied  by  Madsen,**  in  that  it  is  inter- 
mediate, and  only  brought  about  by  the  cooperation  of  two 

*  "Deutsche  med.  Wochenschrift,"  1896,  No.  7. 
f  "Ann.  de  1'Inst.  Pasteur,"  1895.  %  Ibid.,  XH,  1898. 

§  "Berliner  klin.  Wochenschrift,"  1899. 
||  "Trans.  Path.  Soc.  of  London,"  u. 
**  "  Zeitschr.  f.  Hyg.,"  1899,  xxxm,  p.  239. 


Cytotoxic  Serums  121 

factors,  while  the  action  of  the  lysins  of  venom,  the  tetanus 
bacillus,  the  streptococcus,  Bacillus  pyocyaneus,  and  other 
micro-organisms,  is  direct  and  immediate. 

Myers  found,  however,  that  the  hemolytic  substance  of 
venom,  and  Madsen  that  the  hemolytic  products  of  Bacillus 
tetani,  also  produce  reactions  in  animals,  and  that  when  suc- 
cessful immunization  against  them  was  accomplished,  the 
serums  of  the  experiment  animals  became  antidotal  or  in- 
hibiting to  the  action  of  the  respective  lysins. 

Von  Diingern*  found  that  by  injecting  dissociated  epithe- 
lial cells  from  the  trachea  of  oxen  into  the  peritoneal  cavity 
of  guinea-pigs,  it  was  possible  to  produce  epitheliolysins ; 
Lindemann,f  that  emulsions  of  kidney  substance  injected 
into  animals  caused  them  to  form  nephro-lysins  or  nephro- 
toxins ;  Landsteiner  J  and  Metschnikoff  §  in  the  same  manner 
successfully  prepared  spermatoxin  by  injecting  the  sperma- 
tozoa of  one  animal  into  the  peritoneal  cavity  of  another. 
Metalnikoffli  found  that  if  he  introduced  the  spermatozoa  of 
a  guinea-pig  into  the  peritoneum  of  another,  the  spermo- 
toxic  serum  produced  was  solvent  for  the  spermatozoa  of 
both.  Both  Metschnikoff  and  Metalnikoff  also  found  that 
the  spermotoxin  when  introduced  into  animals  was  active 
in  producing  anti-spermotoxin  by  which  the  destructive 
action  of  the  serum  upon  spermatozoa  could  be  inhibited. 

Metschnikoff  **  and  Funck  ff  found  that  animals  treated 
with  emulsions  of  the  spleen,  and  mesenteric  lymph-nodes 
of  one  kind  of  animal,  produced  sera  whose  action  was 
agglutinative  and  solvent  for  leukocytes  and  lymph-cells. 
Delezene  { J  found  that  dissociated  liver  cells  injected  into 
animals  similarly  caused  the  formation  of  a  cytotoxic  serum 
whose  specific  destructive  action  was  upon  them. 

All  of  these  reactions  are  indirect  and  intermediate,  and 
take  place  under  appropriate  conditions  both  in  the  bodies  of 
animals  and  in  the  test-tube. 

But  the  number  of  antigenic  reactions  that  can  be  brought 
about  in  the  bodies  of  animals  seems  to  be  limitless,  and, 
strange  as  it  may  seem,  the  antibodies  produced  in  the  body 

*  "  Miinchener  med.  Wochenschrift,"  1899. 

t  "Ann.  de  1'Inst.  Pasteur,"  1900. 

t  "Centralbl.  f.  Bakt.,"  etc.,  1899,  xxv. 

§  "Ann.  de  1'Inst.  Pasteur,"  1899. 

i|  Ibid.,  1900.  **  Ibid.,  1899- 

ft  "Centralbl.  f.  Bakt.,"  etc.,  xxvn,  1900. 
it  "  Compte  rendu  de  1'Acad.  des  Sciences,"  1900,  cxxx,  pp.  938,  1488. 


122  Immunity 

of  one  animal  may  act  as  antigens  when  introduced  into 
another.  Thus,  Ehrlich  and  Morgenroth  in  their  studies  of 
hemolysis  found  that  serums  rich  in  immune  bodies  produced 
reactions  yielding  anti-immune  bodies,  which  inhibited  the 
activities  of  the  respective  immune  bodies  by  whose  stimula- 
tion they  were  produced. 

The  reactions  which  when  repeated  may  lead  to  immunity 
and  to  the  formation  of  antibodies  seem  to  be  followed  by 
constitutional  disturbances  much  more  profound  than 
would  be  supposed  from  the  apparent  freedom  from  symp- 
toms manifested  by  the  animal.  As  early  as  1839  Magendie 
observed  that  if  a  rabbit  was  given  an  injection  of  albumin, 
and  then,  some  days  later,  a  second  injection,  it  was  made 
very  ill  and  might  die.  About  1900  Mattson  called  the 
author's  attention  in  private  conversation  to  the  fact  that 
when  guinea-pigs  used  for  testing  antitoxic  serums  were  sub- 
sequently injected  with  another  dose  of  serum,  they  com- 
monly died.  Not  being  understood,  the  matter  was  not 
thought  worthy  of  publication.  Otto*  speaks  of  this  fatal 
action  of  serums  as  the  "Theobald-Smith  phenomenon,"  the 
fact  having  first  been  pointed  out  to  him  by  Smith. 

The  first  to  realize  the  importance  of  the  condition  seem 
to  have  been  Portier  and  Richet,|  who  studied  the  effect 
of  extracts  of  the  poisonous  tentacles  of  actiniens  upon 
dogs  which  were  found  to  die  more  quickly  and  from  smaller 
doses  given  at  a  second  injection  than  at  the  first.  To 
this  increase  of  sensitivity  to  the  poison  brought  about  by 
the  initial  dose  they  gave  the  name  anaphylaxis  (av  nega- 
tive, yoXaZts  protection,  destroying  protection  or  breaking 
down  the  defenses). 

The  therapeutic  employment  of  diphtheria  antitoxic  serum 
was  scarcely  popularized  before  the  medical  profession  was 
shocked  by  the   sudden  death   of   the   healthy  child   of   a 
noted  German  professor  after  a  prophylactic  injection,  and 
in  1896  GottsteinJ  was  able  to  collect  8  deaths  following 
the  use  of  the  serum,  4  of  them  being  persons  not  ill  with 
diphtheria.      von    Pirquet  and  Schick§    also   pointed    out 
that  in  a  certain  proportion  of  cases  the  injection  of  horse- 
serum  in  man  is  followed  by  urticarial  eruptions,  joint-pains, 
fever,  swelling  of  the  lymph-bodes,  edema  and  albuminuria, 
*  von  Lenthold,  "Gedenkschrift,"  Bd.  i,  pp.  9,  16,  18. 
t  "Compte  rendu  de  la  Soc.  de  Biol.  de  Paris,"  1902. 
J  "Therap.  Monatschrift,"  1896. 
§  "Die  Serumkrankheit,"  Leipzig  and  Wien,  1905. 


Allergia  or  Anaphylaxis  123 

these  symptoms  usually  appearing  after  an  incubation 
period  of  eight  to  thirteen  days,  and  constituting  what  they 
call  the  "serum  disease,"  or  allergia.  Sometimes  these  re- 
actions are  immediate;  sometimes  death  appears  imminent, 
and,  as  has  been  observed,  death  sometimes  occurs. 

The  investigation  of  the  subject  was  taken  up  in  1905 
by  Rosenau  and  Anderson*,  who  pursued  it  with  great  in- 
terest and  industry,  by  Gay,f  Gay  and  Southard,!  and 
others. 

Experimental  study  shows  that  when  an  animal  is  injected 
with  an  alien  protein  of  almost  any  kind,  a  reaction  takes 
piace  that  usually  is  not  completed  under  six  days.  If  a 
second  injection  is  given  before  the  reaction  is  perfected,  the 
mechanism  of  immunity  is  set  in  action,  and  the  animal  pro- 
ceeds to  defend  itself  through  the  various  means  described. 
If  the  second  administration  be  deferred,  however,  until  the 
first  reaction  is  completed,  it  seems  to  find  the  animal  in  a 
state  of  disturbed  biologic  equilibrium,  the  nature  of  which 
is  not  understood,  but  which  is  characterized  by  a  profound 
disturbance  that  may  terminate  in  death.  The  reaction  is 
quite  specific;  the  sensitization,  once  effected,  may  continue 
throughout  the  remainder  of  the  life  of  the  animal  and  be 
transmitted  from  the  mother  to  her  offspring  through  her 
blood.  The  reaction  can  be  brought  about  by  feeding  the 
protein  or  by  injecting  it.  It  has  an  important  bearing 
upon  infection  and  immunity,  the  chief  example  being  seen 
in  the  tuberculin  reaction. 

The  symptomatology  of  anaphylaxis  is  interesting  and 
characteristic.  When  it  is  desirable  to  study  it,  a  guinea-pig 
is  first  given  a  sensitizing  dose  of  horse-serum.  This  may 
be  very  small.  Rosenau  and  Anderson  found  one  guinea-pig 
to  be  sensitized  by  one-millionth  of  a  cubic  centimeter.  In 
most  of  their  work  they  used  less  than  ^1^  c.c.  It  is  neces- 
sary to  wait  until  the  effects  of  this  first  injection  are  com- 
pletely over  before  giving  the  poisoning  dose.  This  period 
of  incubation  lasts  about  twelve  days.  After  the  lapse  of 
this  time,  the  second  dose,  usually  about  y1^  c.c.,  is  given. 
Both  doses  are  given  by  injection  into  the  peritoneal  cavity. 

*  "Journal  of  Medical  Research,"  1906,  xv,  p.  207;  "Bull.  No.  29 
of  the  Hygienic  Laboratory,"  Washington,  D.  C.,  1906;  "Bull.  No. 
36,"  1907,  Ibid.;  "Jour.  Med.  Research,"  xvi,  No.  3,  p.  381;  "Jour. 
Infectious  Diseases,"  iv,  No.  i,  p.  i,  1907;  "Jour.  Infectious  Diseases," 
vol.  iv,  p.  552,  1907. 

f  "Jour.  Med.  Research,"  May,  1907,  xvi,  No.  2,  p.  143. 

}  Ibid.,  June,  1908,  xvm,  No.  3,  p.  385. 


124  Immunity 

The  symptoms  come  on  almost  immediately.  The 
animal  is  profoundly  depressed,  extremely  uneasy,  pants 
for  breath,  and  suffers  from  intense  itching  of  the  face.  It 
soon  falls,  continues  to  gasp  for  breath,  and  dies  within  an 
hour.  The  disturbances  in  the  body  of  the  animal  are 
sufficient  to  account  for  the  symptoms.  Extensive  lesions 
exist,  the  first  to  be  described  by  Rosenau*  affecting 
the  mucous  membrane  of  the  stomach,  which  appeared 
ecchymotic  and  ulcerated.  Gay  and  Southard  f  found 
hemorrhages  in  most  of  the  organs,  and  believe  anaphy- 
laxis  to  depend  upon  the  presence,  in  the  blood  of  the 
sensitized  animal,  of  a  substance  to  which  they  have  given 
the  name  anaphylactin.  It  seems  difficult,  however,  to 
imagine  how  such  a  substance  could  remain  in  the  blood 
throughout  the  entire  subsequent  life  of  the  animal.  Bes- 
redka  and  Steinhardt  J  found  that  by  the  repeated  injection 
of  horse-serum  into  guinea-pigs,  the  intervals  being  too  short 
to  permit  anaphylaxis,  antianapkylactin  could  be  prepared. 

Anaphylaxis  is  not  a  disturbance  of  the  cells  of  the  body, 
as  some  have  thought,  but  is  at  least  in  part  a  disturbance 
of  the  composition  of  the  blood,  as  can  be  shown  by  the  oc- 
currence of  what  is  known  as  passive  anaphylaxis.  If  the 
blood-serum  of  a  sensitized  animal  be  withdrawn  and  in- 
jected into  a  normal  animal  of  the  same  kind,  it  carries  the 
sensitization  with  it.  The  new  animal,  however,  does  not 
become  sensitized  at  once,  but  only  after  some  days,  hence 
it  is  equally  true  that  the  disturbance  is  not  solely  in  the 
blood,  else  why  should  not  the  sensitization  be  immediately 
present  upon  the  injection  of  the  serum? 

Anaphylaxis  may,  furthermore,  be  local.  Thus,  when 
certain  substances  like  tuberculin  are  dropped  in  the  eye 
there  is  no  effect,  but  when  a  second  application  is  made, 
after  some  weeks,  the  eye  may  be  reddened. 

Vaughan  has  endeavored  to  explain  anaphylaxis  by  assum- 
ing that  when  the  strange  protein  in  the  blood  reaches  the 
cells  it  is  slowly  broken  down  by  enzymic  action,  but  that 
the  cells,  having  once  acquired  the  property  of  destruction, 
seize  eagerly  upon  the  protein  the  next  time  it  is  offered,  dis- 
integrate it  rapidly,  and  so  disseminate  throughout  the  body 

*"Bull.  No.  32  of  the  Hygienic  Laboratory,"  Washington,  D.  C., 
October,  1906. 

t  "Jour.  Med.  Reserach,"  July,  1908,  xix,  No.  i,  pp.  i,  5,  17. 

J  "Ann.  de  1'Inst.  Pasteur,"  xxi,  No.  2,  February  25,  1907,  pp. 
117-127. 


Explanation  of  Immunity  125 

the  degradation  products,  some  of  which  may  be  toxic  and 
account  for  the  reaction. 

Anaphylaxis'may  play  a  role  in  infection.  In  cases  where 
an  attack  of  an  infectious  disease  leaves  no  immunity,  the 
body  may  be  left  hypersensitive  to  subsequent  attacks. 

EXPLANATION  OF  IMMUNITY, 

Before  the  facts  now  at  our  disposal  had  been  gathered 
together,  and  before  the  phenomena  of  immunity  against 
infection  had  been  compared  with  those  of  intoxication, 
Pasteur*  and  Klebsf  endeavored  to  explain  acquired  im- 
munity by  supposing  that  micro-organisms  living  in  the 
infected  animal  used  up  some  substance  essential  to  their 
existence,  and  so  died  out,  leaving  the  soil  unfit  for  further 
occupation.  This  was  known  as  the  "exhaustion  theory." 
WernichJ  and  Chauveau§  thought  it  more  probable  that 
the  micro-organisms  after  having  lived  in  the  body  left 
behind  them  some  substance  inimical  to  their  further 
existence.  This  was  known  as  the  "retention  theory." 
These  hypotheses  are  of  historic  interest  only,  and  deserve 
no  more  than  passing  mention,  as  they  both  fail  to  explain 
natural  immunity  or  immunity  against  intoxication. 

Karl  Roser||  observed  that  the  leukocytes  of  the  bodies  of 
higher  animals  sometimes  enclosed  bacteria  in  their  cyto- 
plasm. Koch,  Sternberg,  and  others  confirmed  the  obser- 
vation, but  no  attention  was  paid  to  it  until  Metschnikoff** 
correlated  it  with  other  known  facts  and  original  observa- 
tions, and  came  to  the  conclusion  that  the  enclosed  bacteria 
had  been  eaten  by  the  leukocytes  in  which  they  were  killed 
and  digested,  and  that  the  behavior  of  the  cells  toward  the 
bacteria  afforded  an  explanation  of  the  mechanism  by  which 
recovery  from  the  infectious  diseases  takes  place.  The 
original  conception  upon  which  this  "theory  of  phagocyto- 
sis" was  founded,  refers  recovery  in  many,  if  not  all  of  the 
infectious  diseases,  to  the  successful  destruction  of  the 
invading  bacteria  by  the  body  cells,  especially  the  leukocytes. 

*  "Compte  rendu  de  la  Soc.  de  Biol  de  Paris,"  xci. 

t  "Arch.  f.  experimentele  Path.  u.  Pharmak.,"  xm. 

I  "Virchow's  Archives,"  Bd.  LXXVIII. 

§  "Compte  rendu  de  la  Soc.  de  Biol.  de  Paris,"  xc  and  xci. 

||  "Beitrage   zur  Biologic  niederster  Organismen,"  Inaugural  Dis- 
sertation, Marburg,  1881. 

**  "Virchow's  Archives,"  Bd.  xcvi,  p.  177;  "Ann.  de  1'Inst.  Pas- 
teur," t.  i,  p.  321,  1887. 


1 26  Immunity 

These  devouring  cells  Metschnikoff  called  phagocytes,  and 
of  them  he  recognized  two  classes,  the  microphages ,  which 
are  white  blood-corpuscles,  and  the  macrop hages,  which  are 
larger  cells  derived  from  the  endothelial  and  other  tissues. 
Metschnikoff,  his  associates,  and  his  pupils  soon  collected 
evidence  sufficient  to  show  that  phagocytosis,  if  not  the 
chief  factor  in  defending  the  body  from  infectious  organisms, 
is  at  least  an  important  one.  Many  of  the  most  interesting 
facts  are  described  in  Metschnikoff 's  books,  "Etudes  sur  Tin 


Fig.  1 8. — Phagocytosis;  the  omentum  immediately  after  injection  of 
typhoid  bacilli  into  a  rabbit.  Meshwork  showing  a  macrophage,  inter- 
mediate forms  and  a  trailer,  all  containing  intact  bacilli  (Buxton  and 
Torry). 

flammation"  and  "Immunite  dans  les  Maladies  Infectieuses," 
which  every  interested  student  of  the  subject  should  read. 

These  studies  show  that  in  nearly  all  cases  in  which 
animals  are  naturally  immune  against  infection,  the  leu- 
kocytes are  active  in  their  phagocytic  behavior  toward 
them;  that  in  acquired  immunity,  the  leukocytes  pre- 
viously inactive,  become  active  toward  them;  that  the 
enclosure  of  bacteria  within  the  cells  sometimes  results  in 
the  death  of  the  cells,  sometimes  in  the  death  of  the  bacteria ; 
that  phagocytosis  is  much  more  active  in  diseases  in  which 
the  bacteria  have  limited  toxicogenic  powers,  and  in  which 
they  probably  exert  a  positively  chemotactic  influence  upon 
the  cells,  than  in  cases  in  which  the  bacteria  are  strongly 
toxicogenic  and  probably  exert  an  injurious  and  negatively 
chemotactic  influence  upon  them,  and  that  when  the  toxico- 
genic power  of  the  bacteria  is  great,  many  of  the  phago- 
cytes are  killed  and  dissolved — phagolysis.  Study  of  the 
primitive  forms  of  animal  life  shows  that  amebae  constantly 


Phagocytosis — Opsonins  127 

feed  upon  smaller  organisms,  some  almost  exclusively  upon 
bacteria,  which  they  are  able  to  kill  and  digest  through  an 
intracellular  enzyme  demonstrated  by  Mouton,*  and  called 
amebadiastase,  and  regarded  as  a  form  of  trypsin.  The 
intracellular  digestion  of  ccelenterate  animals  is  accomplished 
by  means  of  actinodiastase,  an  enzyme  discovered  by 
Fredericq,  and  studied  by  Mesml.  It  seems  to  be  related 
to  papine  and  digests  albuminoids.  The  digestion  of  ery- 
throcytes  and  tissue  fragments  is  accomplished  through  an 
enzyme  of  the  macrophages,  which  Metschnikoff  calls  macro- 
cytase,  that  of  bacteria  through  an  enzyme  of  the  microphages, 
which  he  calls  microcytase.  In  phagolysis  these  respective 
ferments  are  liberated  into  the  plasma,  imparting  to  it  a 
bactericidal  and  bacteriolytic  action  similar  to  that  normally 
peculiar  to  the  cytoplasm  of  the  cells.  The  dissemination 
of  the  enzymes  in  phagolysis,  with  resulting  bacteriolytic 
power  of  the  blood  plasma  and  serum,  is  a  later  modification 
of  the  original  conception  of  Metschnikoff,  that  the  invading 
parasites  were  eaten  up  by  the  phagocytes,  and  was  made 
necessary  by  the  investigation  of  the  bactericidal  property  of 
the  body  juices.  The  experiments  of  Wright  and  Douglasf 
indicate  that  the  action  of  the  phagocytes  upon  the  bacteria 
is  not  immediate,  but  only  subsequent  to  a  preparative 
action  upon  the  organisms  by  substances  contained  in 
serum,  to  which  they  have  given  the  name  "  Opsonins"  (Lat. 
opsono,  "I  prepare  a  meal  for"). 

Long  before  Metschnikoff  began  his  studies  of  the  pha- 
gocytes Traube  and  Gscheidel  I  observed  that  the  blood- 
plasma  possessed  the  power  of  destroying  the  vitality  of 
bacteria.  Grohman§  next  observed  that  not  only  the 
intravascular,  but  also  the  extra  vascular  blood  possessed 
this  property.  The  first  exact  investigations  of  the  subject 
were  made  by  von  Fodor.  ||  The  systematic  investigation  of 
the  bactericidal  activity  of  blood-serum  in  vitro  was  next 
taken  up  by  Fliigge,**  and  more  particularly  by  Nuttall,ft 
who  found  that  different  blood-serums  possessed  the  power 

*  "Compte  rendu  de  1'Acad.  des  Sciences  de  Paris,"  1901,  cxxxm, 
p.  244. 

<  t  "  Proc.  Royal  Society  of  London,"  utxxii,  p.  357,  1904- 
t  "  Jahresberichte  der  Schles.  Ges.  f.  vaterl.  Kultur,"  1874. 
§  "  Untersuchungen  aus  dem  physiol.  Institut  zu  Dorpat,"  Dorpat, 
1884;  Kriiger. 

||  "Centralbl.  f.  Bakt.,"  etc.,  1890,  vn,  p.  753- 
**  "Zeitschrift  fur  Hygiene,"  Bd.  iv,  S.  208. 
ft  Ibid.,  Bd.  IV,  S.  353- 


128  Immunity 

of  killing  bacteria  in  large  numbers,  but  that  the  bactericidal 
power  of  the  serum  soon  disappeared,  after  which  the  serum 
became  a  good  culture-medium  for  the  very  bacteria  it  had 
formerly  destroyed.  Metschnikoff  objected  to  the  observa- 
tions, declaring  that  all  the  phenomena  were  ultimately 
referable  to  the  leukocytes,  so  Nuttall  investigated  pen- 
cardial  fluid  arid  the  aqueous  humor  of  the  eye,  which  were 
also  found  to  possess  bactericidal  powers. 

The  matter  was  next  taken  up  by  Buchner  and  his  as- 
sociates,* who  showed  that  the  blood-plasma  and  blood- 
serum  possessed  exactly  the  same  bactericidal  effects  as  the 
total  blood.  Buchner  and  Nuttall  both  showed  that  the 
exposure  of  the  bactericidal  fluids  to  a  temperature  of  56°  C. 
for  a  few  hours  entirely  destroyed  their  activity,  though  low 
temperatures  were  without  effect  upon  them.  Buchner 
found  that  the  exposure  of  the  serum  to  sunlight  and 
oxygen  also  destroyed  the  bactericidal  power.  Neutraliza- 
tion of  alkaline  serum  did  not  destroy  its  activity,  but 
when  the  serum  was  dialyzed  and  the  NaCl  removed  from 
it,  the  germicidal  power  was  lost,  to  return  again  when  it  was 
restored.  Buchner  called  the  bactericidal  principle  alexin. 

Many  interesting  facts  were  collected  bearing  upon  the 
bactericidal  substance  or  alexin.  Thus  Morof  showed  that 
it  was  proportionally  more  active  in  sucking  infants  than  in 
adults,  and  Ehrlich  and  BriegerJ  found  that  it  passed  from 
mother  to  offspring  in  the  milk. 

At  first  Buchner  regarded  alexin  as  an  albumin,  but  later§ 
he  came  to  look  upon  it  as  a  proteolytic  enzyme,  this  view 
no  doubt  resulting  from  an  endeavor  to  explain  the  relation 
of  alexin  to  immunity  against  intoxication,  in  which  it  was 
necessary  to  show  that  alexin  not  only  killed  bacteria,  but 
also  destroyed  toxins. 

Hankin||  endeavored  to  show  that  there  were  differences 
between  the  substances  destroying  the  bacteria  and  those  act- 
ing upon  their  toxic  products.  To  the  whole  group  he  applied 
the  term  defensive  proteids.  Those  present  in  natural  im- 
munity he  called  sozins,  those  found  in  acquired. immunity 
phylaxins.  Sozins  with  bactericidal  activity  he  further  de- 

*  "  Centralbl.  f  Bakt .,"  etc  ,  1889,  Bd  v,S.  817;  vi,  S.  1;  "Archivfur 
Hygiene,"  1891,  x,  S   727;  "Centralbl.  f.  Bakt  ,"  etc.,  1890,  vn,  S.  76. 
f'Jahresb.  f.  Kinderheilkunde,"  v,  S    396 
t  "Zeitschrift  fur  Hyg.,"  1893,  xm,  S.  336. 
§  "Munch,  med.  Woch.."  1899 
II  "Centralbl.  f.  Bakt.,"  etc.,  xn,  Nos.  22,  23:  xiv.  No.  25. 


Defensive  Proteins,  etc.  129 

scribed  as  mycosozins,  those  with  toxin-destroying  activities 
as  toxosozins.  Phylaxins  with  bactericidal  action  were  called 
mycophylaxins;  those  with  toxin-destroying  properties  toxo- 
phylaxins. 

Metschnikoff  found  it  unnecessary  to  change  his  ideas,  and 
persisted  in  referring  all  the  phenomena  to  the  phagocytes 
or  to  enzymes  derived  from  them. 

At  this  point  it  will  be  evident  to  the  reader  that  the 
phagocytic  theory  and  the  humoral  theory  contain  indubit- 
able evidence  to  show  that  they  are  important  factors  in 
defending  the  body  against  invading  organisms,  and  that  in 
each  we  see  mechanisms  operative  in  certain  cases.  But 
we  have  seen  that  both  Metschnikoff  and  Buchner  are 
obliged  to  strain  a  point  in  order  to  meet  the  requirements 
of  increasing  knowledge  of  the  subject  of  immunity. 

Thus,  when  we  come  to  analyze  Buchner 's  theory  of 
alexins,  we  find  that  if  natural  immunity  depends  upon  the 
ability  of  the  alexins  to  destroy  bacteria,  that  which  takes 
place  in  vitro  should  correspond  with  that  which  takes  place 
in  vivo,  and  that  the  invasion  of  the  animal's  body  by 
bacteria  should  be  accompanied  by  diminution  of  the  bac- 
tericidal substance  in  its  blood,  which  should  be  used  up 
before  the  bacteria  can  be  successful  in  their  invasion. 
Experimental  evidence  is,  however,  at  hand  to  show  that 
this  is  not  always  true. 

Behring  and  Nissen*  found  that  there  was  a  definite 
relation  between  the  bactericidal  power  of  the  blood  in  vitro 
and  the  resisting  powers  of  a  large  number  of  animals 
studied,  but  Lubarschf  showed  the  remarkable  exceptions  of 
the  rabbit,  which  is  highly  susceptible  to  anthrax,  though  its 
blood  is  highly  bactericidal  to  the  anthrax  bacillus,  and  the 
dog,  which  is  scarcely  susceptible  to  anthrax,  though  its 
blood  is  scarcely  bactericidal  to  the  bacillus. 

FluggeJ  found  the  bactericidal  power  of  the  blood  greatly 
lessened  in  thirty-six  hours  after  anthrax  infection,  and 
Nissen  that  a  definite  number  of  bacteria  could  be  killed 
by  a  bactericidal  serum,  after  which  the  alexin  became 
inactive.  The  diminution  of  the  bactericidal  power  was 
shown  to  occur  both  in  the  animal  and  in  the  test-tube.  He 
also  showed  that  the  reactions  of  the  bactericidal  serums 

*  "Zeitschrift  fur  Hygiene,"  1890,  vm,  S.  412. 
f  "Centralbl.  f.  Bakt.,"  etc.,  1889,  vi,  S.  481. 
J  "Zeitschrift  fur  Hygiene,"  iv,  S.  208. 
9 


130  Immunity 

were  specific,  and  that  when  a  culture  of  one  kind  of  bacteria 
was  injected  into  an  animal,  the  immediate  effect  was  to 
diminish  the  activity  of  the  serum  for  that  species,  though 
not  necessarily  for  other  species.  The  diminution  of  bac- 
tericidal energy  was  shown  by  him  to  depend  upon  the 
presence  of  the  bacteria,  as  the  injection  of  filtrates  of  bac- 
terial cultures  did  not  affect  the  bactericidal  properties  of 
the  serum.  This  was  a  very  important  observation. 

There  is  a  correspondence  between  the  behavior  of  the 
phagocytes  and  the  body  juices.  When  the  activity  of  the 
phagocytes  toward  the  bacteria  is  increased,  the  bactericidal 
activity  of  the  serum  is  usually  intensified.  But  immunity 
is  only  partly  explained  by  alexins  and  bacteriolysis,  for 
it  embraces  the  ability  of  the  organism  to  endure  the  effects 
of  toxins  some  of  which  are  in  no  way  connected  with 
bacteria. 

Tolerance  to  certain  toxins  is,  of  course,  natural  to  many 
animals,  and  tolerance  to  usually  destructive  toxins  natural 
to  a  few.  This  toxin-neutralizing  or  annulling  factor 
cannot  be  identical  with  the  bacteria-destroying  mechanism. 
Cobbett,*  Roux  and  Martin,  f  and  BoltonJ  have  shown  that 
horses  that  cannot  be  supposed  ever  to  have  come  into 
contact  with  diphtheria  bacilli,  vary  considerably  in  their 
resistance  to  diphtheria  toxin,  and  that  the  serum  of  the 
resisting  horses  contains  something  that  destroys  or  neu- 
tralizes the  toxin  in  vitro,  as  well  as  exerts  a  protective 
influence  upon  animals  into  which  it  is  injected.  This 
substance  exerts  no  inimical  action  upon  the  diphtheria 
bacilli,  beyond  what  a  normal  serum  would  do,  therefore 
cannot  be  alexin,  but  must  be  antitoxin.  Abel§  found 
that  the  blood  of  healthy  men  occasionally  contained  some 
substance  capable  of  neutralizing  diphtheria  toxin;  Stern 
found  one  normal  serum  capable  of  protecting  against  typhoid 
infection  and  Metschnikoff  one  that  protected  against  cholera 
infection.  Fischel  and  Wunschheim||  found  newly  born 
babies  immune  against  diphtheria,  presumably  because  of 
the  presence  of  a  small  quantity  of  demonstrable  protective 
substance  in  the  blood.  These  are,  however,  peculiar  and 
exceptional  cases. 

*  "Lancet,"  Aug.  5,  1899,  n,  p.  532. 

t  "Ann.  de  1'Inst.  Pasteur,"  vin,  p.  615,  1894. 

t  "Jour,  of  Experimental  Medicine,"  July,  1896,  I,  No.  5. 

§  "Centralbl.  f.  Bakt.,"  etc.,  xvn,  pp.  36,  1895. 

||  "Zeitschr.  fur  Heilkunde,"  1895,  xvi,  p.  429-482. 


The  ''Lateral-chain  Theory"  of  Immunity      131 

The  most  suggestive  and  fascinating  theory  of  immunity 
is  that  of  Ehrlich,  and  is  known  as  the  "  Seitenkettentheorie" 
or  the  "Lateral-Chain  Theory."* 

He  began  his  studies  by  an  investigation  into  the  nature  of 
toxins  and  their  mode  of  action.  The  discovery  that  there 
was  no  constant  relation  between  the  •  intoxicating  and 
antitoxin  combining  powers  of  diphtheria  toxic  bouillon  led 
him  to  the  conclusion  that  the  toxin  molecules  possessed  two 
different  affinities,  which  he  described  as  haptophorous  or 
combining,  and  toxophorous  or  poisoning.  The  former  were 
constant,  the  latter  variable.  The  deterioration  in  the 
strength  of  the  toxic  filtrates  of  bouillon  cultures  of  diph- 
theria bacilli  was  shown  to  depend  upon  the  transformation 
of  the  toxin  into  toxoids  which  were  not  poisonous,  and 
was  shown  to  be  quite  independent  of  the  antitoxin  com- 
bining affinity  of  the  filtrate  which  remained  unaltered. 
The  inevitable  interpretation  seemed  to  be  the  existence 
in  the  bouillon  of  the  haptophorous  and  toxophorous 
groups  described.  Similar  toxophorous  and  haptophorous 
groups  were  shown  to  exist  in  other  toxins — tetanolysin, 
by  Madsen,  venoms,  by  Myers,  and  milk-curdling  fer- 
ments by  Morgenroth.  The  neutralizing  action  of  the 
antibodies  produced  in  the  blood  of  animals  immunized  to 
these  various  substances  depends  upon  the  immediate  and 
direct  combination  or  union  of  haptophorous  groups  in  the 
antibodies  with  corresponding  haptophorous  groups  of  the 
respective  toxins  or  active  bodies. 

The  physiological  activities  of  toxins  differ  from  those  of 
alkaloids  and  other  poisons  differ  in  three  fundamentals: 

*  The  writings  of  Ehrlich  and  his  associates  are  so  numerous  and 
scattered,  and  often  so  fragmentary,  that  instead  of  referring  to  the 
literature  according  to  the  method  adopted  in  other  parts  of  this 
work,  the  reader  who  desires  to  refer  to  the  original  articles  can  best 
do  so  by  making  use  of  the  following:  Ehrlich,  "Die  Werthbemessung 
des  Diphtheric  Heilserums,"  Klinisches  Jahrbuch,  1897;  Ehrlich,  "Die 
Konstitution des  Diphtheriegiftes,"  Deutsche  med.  Woch.,  1898 ;  "Gesam- 
melte  Arbeiten  zur  Irnmunitatsforschung,"  August  Hirschwald,  Berlin, 
1904 — this  work  contains  the  collected  papers  of  Ehrlich  and  his  associ- 
ates, Aschoff,  "  Ehrlich's  Seitenkettentheorie  und  ihre  Anwendung  auf 
die  Kiinst.lichen  Immunusirungs-prozesse,"  Jena,  1902,  and  the  chap- 
ter upon  "Wirkung  und  Entstehung  der  Aktiven  Stoffe  im  Serum 
noch  der  Seitenkettentheorie,"  by  Ehrlich  and  Morgenroth  in  Kolle 
and  Wassermann's  "Handbuch  der  Pathogene  Mikroorganismen," 
Jena,  1904,  Gustav  Fischer.  Readers  unacquainted  with  the  German 
language  may  find  the  essential  facts  in  Ehrlich's  Croonian  Lecture, 
Proceedings  of  the  Royal  Society  of  London,  LXVI,  1900,  p.  424,  and 
in  Welch's  ''Huxley  Lecture,"  Medical  News,  LXXXI,  1902,  2,  p.  721. 


132  Immunity 

first  in  their  ability  to  produce  antibodies  in  the  bodies  of 
animals  into  which  they  are  injected;  second,  the  mani- 
festation of  poisonous  action  only  after  a  definite  incubation 
period,  and,  third,  an  extremely  labile  composition,  by  which 
the  toxin  becomes  quickly  transformed  to  toxoids. 

A  study  of  the  physiological  action  of  toxins  upon  the  cells 
resulted  in  showing  that  certain  definite  specific  affinities 
existed,  and  that  the  union  of  the  toxin  with  the  cell  ante- 
dated the  production  of  symptoms.  In  some  cases  it  was 
even  found  possible  to  disconnect  the  anchored  toxin  by 
bringing  to  the  cells  haptophorous  groups  for  which  the 
haptophorous  elements  of  the  toxin  molecule  were  known 
to  have  an  active  affinity.  Donitz  determined  the  quantity 
of  tetanus  antitoxin  which,  injected  into  the  circulating 
blood  immediately  after  the  toxin,  absolutely  neutralized 

tlsptophiLe  Toxofrhlle  Uaptofrhore        Toxofihore 

group.        group.  group,  g™up.t 


\ 
\ 


Ce 


Fig.  19. — Diagram  to  represent  the  combining  groups  of  the  cell  and 
of  the  toxin  respectively  (after  Ehrlich)  (Hewlett). 

it  and  rendered  all  of  the  circulating  toxin  innocuous.  If  the 
same  quantity  of  antitoxin  was  given  seven  or  eight  minutes 
after  the  injection  of  the  toxin,  death  occurred  from  tetanus, 
exactly  as  if  no  antitoxin  had  been  given.  Evidently  the 
toxin  had  anchored  itself  to  the  nerve-cells  too  quickly  for 
the  antitoxin  to  reach  and  combine  with  it.  Heymans  found 
that  if  an  animal  was  injected  with  tetanus  toxin,  and  its 
entire  blood  withdrawn  immediately  afterward  and  replaced 
by  transfusion,  it  died  of  typical  tetanus  because  in  the  brief 
interval  between  the  toxin  injection  and  the  transfusion,  the 
toxin  molecules  became  anchored  to  the  cell. 

The  ability  of  the  cells  thus  to  anchor  the  toxin  is  supposed 
by  Ehrlich  to  depend  upon  the  existence  of  haptophorous 
combining  affinities,  which  he  describes  as  receptors.  He 
views  the  mode  of  toxin  reception  as  depending  upon  a 


The  "Lateral-chain  Theory"  of  Immunity      133 

mechanism  either  identical  with  or  analogous  to  that  by 
which  cellular  nutrition  is  maintained,  and  points  out  that 
in  the  case  of  methylene-blue  and  other  colored  substances, 
which  afford  an  opportunity  to  make  ocular  observations 
upon  the  absorption  of  the  pigment  by  the  cells,  only  certain 
cells  absorb  the  colors. 

Cell  nutrition  is  therefore  probably  carried  on  through  the 
agency  of  receptors  by  which  appropriate  nutrient  hap- 
tophorous  groups  are  apprehended  and  utilized. 

"We  now  come  to  the  important  question  of  the  signifi- 
cance of  the  toxophile  groups  in  organs.  That  these  are  in 
function  especially  designed  to  seize  on  toxins  cannot  be  for 
one  moment  entertained.  It  would  not  be  reasonable  to 
suppose  that  there  were  present  in  the  organism  many 
hundreds  of  atomic  groups,  destined  to  unite  with  toxins, 
when  the  latter  appeared,  but  in  function  really  playing  no 
part  in  the  processes  of  normal  life,  and  only  arbitrarily 
brought  into  relation  with  them  by  the  will  of  the  investi- 
gator. It  would,  indeed,  be  highly  superfluous,  for  example, 
for  all  our  native  animals  to  possess  in  their  tissues  atomic 
groups  deliberately  adapted  to  unite  with  abrin,  ricin,  and 
crotin,  substances  coming  from  far-distant  tropics." 

"One  may,  therefore,  rightly  assume  that  these  toxophile 
protoplasmic  groups  in  reality  serve  normal  functions  in  the 
animal  organism,  and  that  they  only  incidentally  and  by 
pure  chance  possess  the  capacity  to  anchor  themselves  to 
this  or  that  toxin." 

' '  The  first  thought  suggested  by  this  assumption  was  that 
the  atom  group  referred  to  must  be  concerned  in  tissue 
change ;  and  it  may  be  well  here  to  sketch  roughly  the  laws 
of  cell  metabolism.  Here  we  must,  in  the  first  place,  draw 
a  clear  line  of  distinction  between  those  substances  which 
are  able  to  enter  into  the  composition  of  the  protoplasm,  and 
so  are  really  assimilated,  and  those  which  have  no  such 
capacity.  To  the  first  class  belong  a  portion  of  the  food- 
stuffs, par  excellence;  to  the  second  almost  all  our  pharma- 
cological agents,  alkaloids,  antipyretics,  antiseptics,  etc." 

"How  is  it  possible  to  determine  whether  any  given 
substance  will  be  assimilated  in  the  body  or  not  ?  There  can 
be  no  doubt  that  assimilation  is  in  a  special  sense  a  synthetic 
process — that  is  to  say,  the  molecule  of  the  food-stuff  con- 
cerned enters  into  combination  with  the  protoplasm  by  a 
process  of  condensation  involving  loss  of  a  portion  of  its 


1 34  Immunity 

water.  To  take  the  example  of  sugar,  in  the  union  with 
protoplasm,  not  sugar  itself  as  such,  but  a  portion  of  it, 
comes  into  play,  the  sugar  losing  in  the  union  some  of  its 
characteristic  reactions.  The  sugar  behaves  here  as  it 
does,  e.  g.,  in  the  glucosids,  from  which  it  can  only  be 
obtained  through  the  agency  of  actual  chemical  cleavage. 
The  glucosid  shows  no  traces  of  sugar  when  extracted  in 
indifferent  solvents.  In  a  quite  analogous  manner  the  sugar 
entering  into  the  composition  of  albuminous  bodies  (gly- 
coproteids)  cannot  be  obtained  by  any  method  of  extrac- 
tion; at  least  not  until  chemical  composition  has  previously 
taken  place.  It  is,  therefore,  generally  easy  by  means  of 
extraction  experiments,  to  decide  whether  any  given  com- 
bination in  which  the  cells  take  part  is,  or  is  not,  a  synthetic 
one.  If  alkaloids,  aromatic  amines,  antipyretics,  or  anilin 
dyes  be  introduced  into  the  animal  body,  it  is  an  easy 
matter,  by  means  of  water,  alcohol,  or  acetone,  according  to 
the  nature  of  the  body,  to  remove  all  these  substances  quickly 
and  easily  from  the  tissues." 

"This  is  most  simply  and  convincingly  demonstrated  in 
the  case  of  the  anilin  dyes.  The  nervous  system  stained  with 
methylene-blue  or  the  granules  of  the  cells  stained  with 
neutral  red  at  once  yield  up  the  dye  in  the  presence  of  alco- 
hol. We  are,  therefore,  obliged  to  conclude  that  none  of 
the  foreign  bodies  just  mentioned  enter  synthetically  into 
the  cell  complex;  but  are  merely  contained  in  the  cells  in 
their  free  state."  ....  "Hence  with  regard  to  the 
pharmacologically  active  bodies  in  general,  it  is  not  allow- 
able to  assume  that  they  possess  definite  atom  groups, 
which  enter  into  combination  with  corresponding  groups 
of  the  protoplasm.  This  corresponds,  as  I  may  remark 
beforehand,  with  the  incapacity  of  all  these  substances  to 
produce  antitoxins  in  the  animal  body.  We  must,  therefore, 
conclude  that  only  certain  substances,  food-stuffs,  par 
excellence,  are  endowed  with  properties  admitting  of  their 
being,  in  the  previously  defined  sense,  chemically  bound  by 
the  cells  of  the  organism.  We  are  obliged  to  adopt  the  view 
that  the  protoplasm  is  equipped  with  certain  atomic  groups, 
whose  function  especially  consists  in  fixing  to  themselves 
certain  food- stuffs  of  importance  to  the  cell-life."  We  may 
assume  that  the  protoplasm  consists  of  a  special  executive 
center,  in  connection  with  which  are  nutritive  side-chains, 
which  possess  a  certain  degree  of  independence  and  which 


The  "Lateral-chain  Theory"  of  Immunity      135 

may  differ  from  one  another  according  to  the  requirements 
of  the  different  cells.  And  as  these  side-chains  have  the 
office  of  attaching  to  themselves  certain  food-stuffs,  we  must 
also  assume  an  atom-grouping  in  these  food-stuffs  them- 
selves, every  group  uniting  with  a  corresponding  combining 
group  of  a  side-chain.  The  relationship  of  the  correspond- 
ing groups,  i.  c.,  those  of  the  food-stuff,  and  those  of  the  cell, 
must  be  specific.  They  must  be  adapted  to  one  another, 
as,  e.  g.,  male  and  female  screw  (Pasteur),  or  as  lock  and 
key  (E.  Fischer).  From  this  point  of  view,  we  must  con- 
template the  relation  of  the  toxin  to  the  cell." 

"We  have  already  shown  that  the  toxins  possess  for  the 
antitoxins  an  attaching  haptophore  group,  which  accords 
entirely  in  its  nature  with  the  conditions  we  have  ascribed 
to  the  relation  existing  between  the  food-stuffs  and  the  cell 
side-chains.  And  the  relation  between  toxin  and  cell  ceases 
to  be  shrouded  in  mystery  if  we  adopt  the  view  that  the 
haptophore  groups  of  the  toxins  are  molecular  groups  fitted 
to  unite  not  only  with  the  antitoxins,  but  also  with  the  side- 
chains  of  the  cells,  and  that  it  is  by  their  agency  that  the 
toxin  becomes  anchored  to  the  cells." 

' '  We  do  not,  however,  require  to  suppose  that  the  side- 
chains,  which  fit  the  haptophore  group  of  the  toxins,  that 
is,  the  side-chains  which  are  toxophile,  represent  some- 
thing having  no  function  in  the  normal  cell  economy.  On 
the  contrary,  there  is  sufficient  evidence  that  the  toxophile 
side-chains  are  the  same  as  those  which  have  to  do  with  the 
taking  up  of  the  food-stuffs  by  the  protoplasm.  The 
toxins  are,  in  opposition  to  other  poisons,  of  extremely 
complex  structure,  standing  in  their  origin  and  chemical 
constitution  in  very  close  relationship  to  the  proteids  and 
their  nearest  derivatives.  It  is,  therefore,  not  surprising 
that  they  possess  a  haptophore  group  corresponding  with 
that  of  a  food-stuff.  Alongside  of  the  binding  haptophore 
group,  which  conditions  their  union  to  the  protoplasm,  the 
toxins  are  possessed  of  a  second  group,  which  in  regard  to 
the  cell  is  not  only  useless,  but  actually  injurious.  And  we 
remember  that  in  the  case  of  the  diphtheria  toxin  there  was 
reason  to  believe  that  there  existed  alongside  of  the  hapto- 
phore group  another  and  absolutely  independent  toxophore 
group."  .  .  .  .  "As  has  been  said,  the  possession  of  a 
toxophile  group  by  the  cell  is  the  necessary  preliminary 
and  cause  of  the  poisonous  action  of  the  toxin."  .... 


136  Immunity 

"If  the  cells  of  these  organs  [organs  essential  to  life]  lack 
side-chains  fitted  to  unite  with  them,  the  toxophore  group 
cannot  become  fixed  to  the  cell,  which  therefore  suffers  no 
injury,  i.  e.,  the  organism  is  naturally  immune.  One  of  the 
most  important  forms  of  natural  immunity  is  based  upon 
the  circumstance  that  in  certain  animals  the  organs  essen- 
tial to  life  are  lacking  in  those  haptophore  groups  which 
seize  upon  definite  toxins.  If,  for  example,  the  ptomaine 
occurring  in  sausages,  which  for  man,  monkeys,  and  rabbits 
is  toxic  in  excessively  minute  doses,  is  for  the  dog  harm- 
less in  quite  large  quantities,  this  is  because  the  binding 
haptophore  groups  being  wanting,  the  ptomaine  cannot, 
in  the  dog,  enter  into  direct  relation  with  organs  essential 
to  life."  .  .  .  "The  haptophore  group  exercises  its 
activity  immediately  after  injection  into  the  organism,  while 


Fig.  20. — Shows  how  the  haptophores  having  united,  the  toxophores 
find  a  secondary  adaptation  to  the  cell,  and  so  can  poison  it  (after 
Ehrlich)  (Hewlett). 

in  all  toxins — with  the  perhaps  solitary  exception  of  snake- 
venom — the  toxophore  group  comes  into  activity  after  the 
lapse  of  a  longer  or  shorter  incubation  period  which  may, 
e.  <?.,  in  the  case  of  diphtheria  toxin,  extend  to  several  weeks." 
"The  theory  above  developed  allows  of  an  easy  and  natural 
explanation  of  the  origin  of  antitoxins.  In  keeping  with 
what  has  already  been  said,  the  first  stage  in  the  toxin 
action  must  be  regarded  as  the  union  of  the  toxin  by  means 
of  its  haptophore  group  to  certain  'side-chains'  of  the  cell 
protoplasm.  This  union  is,  as  animal  experiments  with  a 
great  number  of  toxins  show,  a  firm  and  enduring  one.  The 
side-chain  involved,  so  long  as  the  union  lasts,  cannot  exer- 
cise its  normal  nutritive  physiological  function — the  taking 
up  of  food-stuffs.  It  is,  as  it  were,  shut  out  from  participat- 
ing, in  the  physiological  sense,  in  the  life  of  the  cell.  We 


The  "Lateral-chain  Theory"  of  Immunity      137 

are,  therefore,  now  concerned  with  a  defect  which,  according 
to  the  principles  so  ably  worked  out  by  Professor  Carl 
Weigert,  is  repaired  by  regeneration.  These  principles,  in 
fact,  constitute  the  leading  conception  of  my  theory.  If 
after  union  has  taken  place,  new  quantities  of  toxin  are 
administered  at  suitable  intervals  and  in  suitable  quantities, 
the  side-chains,  which  have  been  reproduced  by  the  regen- 
erative process,  are  taken  up  anew  into  union  with  .the 
toxin,  and  so  again  the  process  of  regeneration  gives  rise  to 
the  formation  of  fresh  side-chains.  In  the  course  of  the 
progress  of  typical  systematic  immunization,  as  this  is 
practised  in  the  case  of  diphtheria  and  tetanus  toxin  espe- 
cially, the  cells  become,  so  to  say,  educated  or  trained  to 
reproduce  the  necessary  side-chains  in  ever-increasing 


Fig.  2 1 . — Cells  with  various  receptors  or  haptophorous  groups  of  the 
first  order  (a),  adapted  to  combination  with  the  haptophorous  groups  (6) 
of  various  chemical  compounds  brought  to  them.  It  will  be  noted  that 
there  is  no  mechanism  by  which  the  toxDphorous  elements  of  the  mole- 
cules (c)  can  be  brought  to  the  cell. 


quantity.  As  Weigert  has  confirmed  by  many  examples, 
this,  however,  does  not  take  place  by  the  simple  replacement 
of  the  defect;  the  compensation  proceeds  far  beyond  the 
necessary  limit;  indeed,  overcompensation  is  the  rule. 
Thus  the  lasting  and  ever-increasing  regeneration  must 
finally  reach  a  stage  at  which  such  an  excess  of  side-chains 
is  produced  that,  to  use  a  trivial  expression,  the  side-chains 
are  present  in  too  great  a  quantity  for  the  cell  to  carry  and 
are,  after  the  manner  of  a  secretion,  handed  over  as  needless 
ballast  to  the  blood.  Regarded  in  accordance  with  this 
conception,  the  antitoxins  represent  nothing  more  than  side- 
chains  reproduced  in  excess  during  regeneration  and  therefore 
pushed  off  from  the  protoplasm  and  so  coming  to  exist  in  the 
free  state," 


138 


Immunity 


The  essence  of  Ehrlich's  theory  is  tersely  expressed  by 
Behring:  "The  same  substance  which  when  incorporated 
in  the  cells  of  the  living  body,  is  the  prerequisite  and  con- 
dition for  an  intoxication,  becomes  the  means  of  cure  when 
it  exists  in  the  circulating  blood." 


Figs.    22  and  23. — Show  the  regeneration  of  the  cell-haptophores  or 
receptors  to  compensate  for  the  loss  of  those  thrown  out  of  service. 


Fig.  24. — Shows  the  number 
of  haptophores  regenerated  by 
the  cell  becoming  excessive,  they 
are  thrown  off  into  the  tissue 
juice. 


Fig.  25. — Explains  what  anti- 
toxins are  and  how  they  are 
formed.  The  liberated  receptors 
in  the  tissue  juice  and  in  the 
blood,  possess  identical  combin- 
ing affinities  with  those  upon  the 
cell,  and  meeting  the  adapted 
haptophorous  elements  in  the 
blood,  combine  with  them,  thus 
keeping  them  from  the  cells. 


Continuing  the  quotations  from  Ehrlich's  Croonian  Lec- 
ture, we  find:  "In  the  first  place,  our  theory  affords  an 
explanation  of  the  specific  nature  of  the  antitoxins,  that 
tetanus  antitoxin  is  only  caused  to  be  produced  by  tetanus 
toxin,  and  diphtheria  antitoxin  through  diphtheria  toxin. 


The  "Lateral-chain  Theory"  of  Immunity      139 

This  very  specific  nature  of  the  affinity  between  toxin  and 
cell  is  the  necessary  preliminary  and  cause  of  the  toxicity 
itself.  Further,  our  theory  makes  it  easy  to  understand  the 
long-lasting  character  of  the  immunity  produced  by  one  or 
several  administrations  of  toxin,  and  also  the  fact  that  the 
organism  reacts  to  relatively  small  quantities  of  toxin  by 
the  production  of  very  much  greater  quantities  of  anti- 
toxin." By  the  act  of  immunization,  certain  cells  of  the 
organism  become  converted  into  cells  secreting  antitoxin  at 
the  same  rate  as  this  is  excreted.  New  quantities  of  anti- 
toxin are  constantly  produced  and  so  throughout  a  long 
period  the  antitoxin  content  of  the  serum  remains  nearly 
constant.  The  secretory  nature  of  the  formation  of  anti- 
toxins has  been  very  strikingly  illustrated  by  the  beautiful 
experiments  of  Salmonson  and  Madsen,  who  have  shown 
that  pilocarpine,  which  augments  the  secretion  of  most 
glands,  also  occasions  in  immunized  animals  a  rapid  increase 
in  the  antitoxin  content  of  the  serum." 

"The  production  of  antitoxins  must,  in  keeping  with  our 
theory,  be  regarded  as  a  function  of  the  haptophore  group  of 
the  toxin,  and  it  is  easy  therefore  to  understand  why,  out  of 
the  great  number  of  alkaloids,  none  are  in  a  position  to 
cause  the  production  of  antitoxins.  Conversely,  indeed, 
I  recognize  in  this  incapacity  of  the  alkaloids,  in  opposition 
to  the  toxins,  to  produce  antitoxins,  a  further  and  salient 
proof  of  the  truth  of  the  deduction  I  have  previously  based 
on  chemical  grounds,  that  the  alkaloids  possess  no  haptophore 
group  which  anchors  them  to  the  cells  of  organs.  To  formu- 
late a  general  statement,  the  capacity  of  a  body  to"  cause  the 
production  of  antitoxin  stands  in  inseparable  connection 
with  the  presence  of  a  haptophore  atomic  group.  In  the 
formation  of  antitoxin  the  toxophore  group  of  the  toxin 
molecule  is,  on  the  contrary,  of  absolutely  no  moment.  But 
the  toxoid  modification  of  the  toxins,  in  which  the  hapto- 
phore group  of  the  toxin  is  retained,  while  the  toxophore 
group  has  ceased  to  be  active,  possesses  the  property  of  pro- 
ducing antitoxins.  Indeed,  in  some  cases  of  extremely 
susceptible  animals,  immunity  can  only  be  attained  by 
means  of  the  toxoid s,  and  not  by  the  too  strongly  acting 
toxins."  ....  "The  symptoms  of  illness  due  to  the 
action  of  the  toxophore  group,  therefore,  play  no  part  in  the 
production  of  antitoxin."  The  effect  of  enzymes  upon  the 
organism  with  the  production  of  an ti- bodies,  and  the  "specific 


140  Immunity 

precipitins"  caused  by  the  injection  of  milk,  albumin,  and 
peptones  into  animals  may  be  looked  upon  as  "having  their 
origin  in  the  most  widely  diverse  organs,  and  representing 
nothing  more  than  nutritive  side-chains,  which,  in  the  course 
of  the  normal  nutritive  processes  have  been  developed  in 
excess  and  pushed  off  into  the  blood." 

' '  Much  more  complex  than  in  the  cases  hitherto  discussed 
are  the  conditions  when,  instead  of  the  relatively  simple 
metabolic  products  of  microbes,  the  living  micro-organisms 
themselves  come  to  be  considered,  as  in  immunization  against 
cholera,  typhoid,  anthrax,  swine-fever,  and  many  other  in- 
fectious diseases.  Thus  there  come  into  existence  alongside 
of  the  antitoxins  produced  as  a  result  of  the  action  of  the 
toxins,  manifold  other  reaction  products.  This  is  because 
the  bacterium  is  a  highly  complicated  living  cell  of  which 
the  solution  in  the  organism  yields  a  great  number  of  bodies 
of  different  nature,  in  consequence  of  which  a  multitude  of 
'antikorper'  are  called  into  existence.  Thus  we  see,  as  a 
result  of  the  injection  of  bacterial  cultures,  that  there  arise 
alongside  of  the  specific  bacteriolysins,  which  dissolve  the 
bacteria,  other  products,  as,  for  example,  the  'coagulins' 
(Kraus,  Bordet),  i.  £.,  substances  which  are  able  to  cause 
the  precipitation  of  certain  albuminous  bodies  contained  in 
the  culture  fluid  injected;  also  the  much-discussed  agglu- 
tinins  (Durham,  Gruber,  Pfeiffer),  the  antiferments  (von 
Dungern),  and  no  doubt  many  other  bodies  which  have  not 
yet  been  recognized.  It  is  by  no  means  unlikely  that  each 
of  these  reaction  products  finds  its  origin  in  special  cells  of 
the  body;  on  the  other  hand,  it  is  quite  likely  that  the  for- 
mation of  any  single  one  of  these  bodies  is  not  of  itself 
sufficient  to  confer  immunity.  Thus,  in  the  case  of  the  in- 
troduction of  bacteria  into  the  body  we  have  to  do  with  a 
many-sided  production  of  different  forms  of  'antikorper,' 
each  of  which  is  directed  only  against  one  definite  quality 
or  metabolic  product  of  the  bacterial  cell.  Accordingly, 
in  recent  times,  the  practice  of  using  for  the  production  of 
immunization  definite  toxic  bodies  isolated  from  the  bacterial 
cells  has  been  more  and  more  given  up,  and  for  this  purpose 
it  is  now  regarded  as  important  to  employ  the  bacterial  cells 
as  intact  as  possible."  ....  "The  most  interesting 
and  important  substances  arising  during  such  an  immuniz- 
ing process  are  without  doubt  the  bacteriolysins."  .... 
"Belfanti  and  Carbone  first  discovered  the  remarkable  fact 


The  "  Lateral-chain  Theory"  of  Immunity      141 


that  horses  which  had  been  treated  with  the  blood-corpuscles 
of  rabbits  contain  in  their  serum  constituents  which  are 
poisonous  for  the  rabbit,  and  for  the  rabbit  only."  .  .  . 
"Bordet  showed  shortly  thereafter  that  in  the  case  quoted 
there  was  present  in  the  serum  a  specific  hemolysin  which 
dissolved  the  corpuscles  of  the  rabbit.  He  also  proved  that 
these  hemolysins — as  had  already  been  shown  by  Buchner 
and  Daremberg  in  the  case  of  similarly  acting  bodies  which 
are  present  in  normal  blood — lost  their  solvent  property  on 
being  maintained  during  half  an  hour  at  a  temperature  of 
55°  C.  Bordet  added,  further,  a  new 
fact,  that  the  blood-solvent  property 
of  those  sera  which  had  been  deprived 
of  solvent  power  by  heat,  the  solvent 
action  could  be  restored  if  certain  nor- 
mal sera  were  added  to  them.  By  this 
important  observation  an  exact  analogy 
was  established  with  the  facts  of  bacteri- 
olysis as  elicited  by  the  work  of  Pfeiffer, 
Metschnikoff,  and  Bordet."  .... 

"In  collaboration  with  Dr. 
Morgenroth,  I  have  sought  in  regard  to 
this  question,  for  which  hemolysis  offered 
prospects  favorable  to  experimentation, 
to  make  clear  the  mechanism  concerned 
in  the  action  of  these  two  compounds — 
the  stable,  which  may  be  designated 
'immune  body/  and  the  unstable,  which 
may  be  designated  'complement' — which 
acting  together  effect  the  solution  of  the 
red  blood-corpuscles.  For  this  purpose, 
in  the  first  place,  solutions  containing 
either  only  the  'immune  body  '  or  only  the  'complement' 
were  brought  in  contact  with  suitable  blood-corpuscles,  and 
after  separation  of  the  fluid  and  the  corpuscles  by  centrifu- 
galization,  we  investigated  whether  these  substances  had 
been  taken  up  by  the  red  corpuscles  or  remained  behind  in 
the  fluid.  The  proof  of  its  location  in  the  one  position  or  in 
the  other  was  readily  forthcoming,  since  to  restore  the 
hemolysin  to  its  former  activity,  it  was  only  necessary  to 
add  to  the  'immune  body'  a  fresh  supply  of  'complement,' 
or  to  the  'complement'  a  fresh  supply  of  'immune  body' 
in  order  that  the  presence  of  the  hemolysin  in  its  integrity 


Fig.  26. — Com- 
bination of  cell  (a), 
amboceptor  (6), 
and  complement 
(c) .  The  ambo- 
ceptor may  unite 
with  the  cell,  but 
cannot  affect  it 
alone.  The  com- 
plement cannot 
unite  with  the  cell 
except  through  the 
amboceptor,  hav- 
ing no  adaptation 
to  the  cell  directly. 


142  Immunity 

might  be  shown  by  the  occurrence  of  solution  of  the  red 
cells.  The  experiments  proved  that,  after  centrifugalizing, 
the  'immune  body'  is  quantitatively  bound  to  the  red  blood- 
corpuscles,  and  that  the  'complement/  on  the  contrary, 
remains  entirely  behind  in  the  fluid.  The  presence  of  the 
two  components  in  contact  with  blood-corpuscles  only 
occasions  the  solution  of  these  at  higher  temperatures,  and 
not  at  o°  C.  And  an  active  hemolytic  serum  (with  'immune 
body'  and  'complement'  both  present)  having  been  placed 
in  contact  with  red  blood-corpuscles  and  maintained  for  a 
while  at  o°  C.,  it  was  found  after  centrifugalizing  that,  under 
these  circumstances  also  the  'immune  body'  had  united  with 
the  red  blood-corpuscles,  but  that  the  'complement'  re- 
mained in  the  serum.  This  experiment  showed  that  both 
components  must,  at  a  temperature  of  o°  C.,  have  existed 
alongside  of  one  another  in  a  free  condition."  .... 

"But  when  analogous  experiments  were  undertaken  at  a 
higher  temperature  it  was  found  that  both  components  were 
retained  in  the  sediment. 

"These  facts  can  only  be  explained  by  making  certain 
assumptions  regarding  the  constitution  of  the  two  com- 
ponents, i.  <?.,  of  the  'immune  body'  and  the  'complement.' 
In  the  first  place,  two  haptophore  groups  must  be  ascribed 
to  the  'immune  body,'  one  having  affinity  for  a  correspond- 
ing haptophore  group  of  the  red  blood-corpuscles  and  with 
which  at  a  lower  temperature  it  quickly  unites,  and  another 
haptophore  group  of  a  lesser  chemical  affinity,  which  at  a 
higher  temperature  becomes  united  with  the  'complement' 
present  in  the  serum.  Therefore  at  the  higher  temperature 
the  red  blood-corpuscles  will  draw  to  themselves  those 
molecules  of  the  'immune  body'  which  in  the  fluid  have 
previously  become  united  to  the  'complement.'  In  this 
case  the  'immune  body'  represents  in  a  measure  the  connect- 
ing chain  which  binds  the  complement  to  the  red  blood- 
corpuscles  and  so  brings  them  under  its  deleterious  influence. 
Since  under  the  influence  of  the  'complement' — at  least,  in 
the  case  of  the  bacteria — appearances  are  to  be  observed 
(for  example,  in  the  PfeifTer  phenomenon)  which  must  be 
.regarded  as  analogous  to  digestion,  we  shall  not  seriously 
err  if  we  ascribe  to  this  'complement'  a  ferment-like  char- 
acter." .  .  .  .  "Having  obtained  a  precise  conception 
of  the  method  of  action  of  the  lysins  of  the  serum — of  the 
hemolysins,  and  thereby  also  of  the  bacteriolysins — it  be- 


The  "Lateral-chain  Theory"  of  Immunity      143 


comes  possible  for  us  to  attempt  to  solve  the  mystery  of  the 
origin  of  these  bodies.  I  have  in  the  beginning  of  this 
lecture*  fully  developed  the  'side-chain  theory,'  according 
to  which  the  antitoxins  are  merely  certain  of  the  protoplasm 
4  side-chain'  which  have  been  produced  in  excess  and  pushed 
off  into  the  blood. 

"The  toxins  as  secretion  products  of  the  cells  are  in  all 
likelihood  still  relatively  uncomplicated  bodies;  at  least  by 
comparison  with  the  primary  and  complex  albumins  of  which 
the  living  cell  is  composed. 

"If  we  now  recognize  that  the  different  lysins  arise  only 
through  absorption  of  highly  com- 
plex cell  material — such  as  red  blood- 
corpuscles  or  bacteria — then  the  ex- 
planation, in  accordance  with  what 
I  have  said,  is  that  there  are  present 
in  the  organism  'side-chains'  of  a 
special  nature,  so  constituted  that 
they  are  endowed  not  only  with  an 
atomic  group  by  virtue  of  the  affini- 
ties of  which  they  are  enabled  to  pick 
up  material,  but  also  with  a  second 
atomic  group,  which,  being  ferment- 
loving  in  its  nature,  brings  about  the 
digestion  of  the  material  taken  up. 
Should  the  pushing  off  of  these  'side- 
chains  '  be  forced,  as  it  were,  by  im- 
munization, then  the  'side-chains' 
thus  set  free  must  possess  both  groups, 
and  will,  therefore,  in  their  character- 
istics entirely  correspond  with  what 

we  have  placed  beyond  doubt  as  regards  the  'immune 
body'  of  the  hemolysin." 

An  analysis  of  this  theory  shows  complete  natural  im- 
munity to  depend  upon  the  absence  of  haptophore  groups 
(receptors)  by  which  the  toxins  can  be  united  to  the  cells. 
Extreme  sensitivity  or  susceptibility  probably  depends 
upon  the  adapted  haptophores  being  present  or  at  least 
most  numerous  upon  the  cells  of  highly  vital  organs ;  com- 
parative insensitivity  or  insusceptibility  upon  the  fact  that 

*  These  lengthy  extracts,  through  which  I  have  endeavored  to 
enable  Ehrlich  to  explain  his  theory  to  the  reader,  are  taken  from  his 
"  Croonian  Lecture,"  delivered  before  the  Royal  Society  of  London, 
March  22,  1900. 


Fig.  27. — Cell  with 
receptors  of  the  second 
order  (a)  by  which  the 
cells  fix  useful  mole- 
cules, of  albumins,  etc., 
on  one  hand  (6),  and  zy- 
mogen  molecules  (c)  on 
the  other  hand,  and 
make  use  of  the  one  sub- 
stance through  the  ac- 
tion of  the  other. 


144  Immunity 

the  greater  number  of  haptophore  groups  are  attached  to 
comparatively  unimportant  cells  whose  combining  affinities 
have  to  be  satisfied  before  combination  with  more  vital  cells 
can  be  accomplished.  In  some  cases  natural  immunity  is 
increased  by  the  presence  of  free  haptophore  groups  (anti- 
toxin) in  the  blood. 

Acquired  immunity  against  toxins  depends  upon  the 
regeneration  of  the  cellular  haptophores  or  receptors  which, 
being  liberated  into  the  body  juices,  fix  the  haptophores  of 
the  toxin  molecules  before  they  are  able  to  reach  the  cells 
themselves.  Antitoxins  and  other  anti-bodies,  including  the 
lysins,  consist  of  liberated  cellular  haptophores  or  receptors, 
the  former  having  a  single  combining  affinity,  the  latter  a 
double  combining  affinity,  by  which  they  unite,  on  the  one 
hand,  with  the  cell  to  be  dissolved,  on  the  other  with  the 
complement  by  which  it  is  to  be  dissolved.  Anti-bodies 
having  this  double  combining  affinity  have  been  called 
' '  amboceptors"  by  Ehrlich.  They  are  variously  known  in 
different  writings  as  "immune  bodies,"  amboceptors,  sub- 
stance sensibilisatrice,  desmon,  and  fixateur.  The  "comple- 
ment" or  "addiment"  of  Ehrlich  is  also  called  alexin  and 
cytase.  Khrlich  conceives  every  amboceptor  and  every 
complement  to  be  specific,  but  Bordet  and  others,  while 
admitting  that  the  amboceptor  is  specific,  hold  that  there  is 
but  one  complement  or  cytase. 

It  has  already  been  shown  that  MetschnikofFs  primitive 
conception  of  phagocytosis,  that  of  the  body  being  defended 
against  the  invasive  power  of  the  microparasites  by  the 
incorporation  and  digestion  of  the  microparasites,  had  to 
be  modified  to  conform  to  the  increasing  information  upon 
the  immunity  reactions.  He  has  persistently  clung  to  the 
idea  that  the  phagocytes  are  the  essential  factors,  but  has 
changed  the  conception  of  "phagocytosis"  to  make  it 
applicable  to  the  condition  shown  by  advancing  knowledge. 
When  invasive  organisms  enter  the  body,  positive  chemotac- 
tic  influences  determine  that  they  are  met  by  a  sufficient  num- 
ber of  phagocytes  to  devour  and  destroy  them.  If  the  invad- 
ing organisms  are  too  powerful  and  the  phagocytes  are  killed 
by  them  and  their  toxins,  the  phagolysis  or  dissolution  of  the 
phagocytes  liberates  their  enzymes  into  the  blood.  These 
liberated  enzymes  now  act  deleteriously  upon  the  invaders, 
tending  to  agglutinate — aggregate  them  in  clumps — and 
sensitize  them  to  the  future  action  of  the  phagocytes  by 
which  they  may  be  taken  up.  Thus,  the  blood-serum  ac- 


The  "Lateral-chain  Theory"  of  Immunity      145 

quires  a  "fixing"  or  "sensitizing"  quality  from  the  presence 
of  the  "fixateur"  or  "substance  sensibilisatrice."  The 
actual  solution  of  the  bacteria  is  accomplished  by  the 
"microcytase"  of  the  leukocytic  phagocytes.  Thus,  we 
find  that  Metschnikoff  is  prepared  to  account  for  the  "am- 
boceptor"  or  "immune  body"  of  Ehrlich,  which  is  the 
fixateur,  and  the  "complement,"  which  is  the  " microcytase." 
When  the  phagolysis  is  excessive,  either  from  infection  or 
intoxication,  both  the  fixateur  and  microcytase  are  free  in 
the  serum.  In  cases  where  the  bacteria  exert  a  negatively 
chemotactic  influence  upon  the  leukocytes,  no  immunity 
exists.  The  reactive  phenomena  occasioned  by  the  intro- 
duction of  tissue  elements  and  blood-corpuscles,  depend  upon 
enzymes  derived  from  the  macrophages,  the  "fixateur"  as 
before,  and  "macrocytase." 

The  antitoxins  are  similarly  accounted  for:  the  cellular 
digestive  enzymes  exert  their  action  not  only  upon  the 
organized  complex  molecules  of  the  microparasites,  but  also 
upon  their  more  simple  toxic  products,  fixing  or  otherwise 
altering  them  until  they  can  be  finally  destroyed. 

It  will  thus  be  seen  that  the  two  chief  theories,  though  they 
appear  discordant  v/hen  explained  independently  of  one 
another,  are  fairly  well  coordinated.  Ehrlich  believes  the 
active  bodies  to  be  the  products  of  those  cells  of  the  body 
for  whose  haptophorous  combining  groups  the  haptophorous 
groups  of  the  active  bodies  engaged,  and  does  not  attribute 
the  function  to  any  particular  group  of  cells ;  Metschnikoff 
attributes  all  the  activities  to  the  phagocytes,  and  especially 
the  leukocytes.  Ehrlich  looks  upon  the  phenomena  as 
chemical  and  pictures  them  as  taking  places  independently 
of  the  cells;  Metschnikoff  looks  upon  them  as  vital  and 
brought  about  by  the  agency  of  living  cells. 

The  fundamental  ideas  embodied  in  the  "lateral-chain  theory"  of 
immunity  may,  by  reversing  the  hypothesis  and  considering  the  bacte- 
rial instead  of  the'  body  cells  to  be  upon  the  defensive,  be  made  to  ex- 
plain other  phenomena  of  immunity.  Walker*  seems  to  have  been 
the  pioneer  in  this  field,  and  his  researches  show  that  it  is  possible  to 
immunize  bacteria  against  "immune  serums"  by  cultivating  them  in 
media  containing  increasing  proportions  of  the  immune  serums.  The 
bacteria  thus  cultivated  were  of  increased  virulence.  The  idea  was 
further  amplified  by  Welch  in  his  Huxley  Lecture. f  The  micro- 
organismal  cells  must  be  regarded  as  endowed  with  receptors  of  their 
own,  fitted  for  combination  with  adapted  haptophorous  elements  in  the 
juices  reaching  them,  and  therefore  capable  of  reacting  toward  such 

*"Jour.  of  Path,  and  Bact,"  vm,  No.  1,  p.  34,  March,  1902. 
f" British    Medical   Journal,"    Oct.    11,    1902,  p.    1105;    "Medical 
News,"  Oct.  18,  1902. 


146  Immunity 

substances  exactly  as  do  the  cells  of  the  host.  As  the  host  reacts 
toward  the  active  products  of  the  bacteria,  so  the  bacteria  react  toward 
the  defensive  products  of  the  host,  and  as  the  cells  of  the  former  are 
stimulated  to  the  production  of  immune  bodies  that  shall  facilitate 
bacteriolysis,  so  the  latter  are  stimulated  to  antagonize  their  action  by 
producing  neutralizing  bodies.  These  neutralizing  bodies  by  which 
the  defenses  of  the  host  are  broken  down  are  among  those  described 
by  Bail*  as  " aggressins." 

Thus,  as  the  cells  of  the  host  invaded  are  constantly  reacting  to  the 
active  bodies  produced  by  the  invading  parasites,  so  the  latter  are  re- 
acting toward  the  defensive  products  of  the  former.  If  the  reactive 
processes  of  the  host  predominate,  immunity  and  the  destruction  of 
the  parasites  result;  if  those  of  the  bacteria  predominate,  increased 
virulence,  facilitated  invasion,  and  death  of  the  host  may  result.  This 
hypothesis  also  serves  to  make  clear  why  micro-organisms  entering  the 
body  not  infrequently  show  a  marked  tendency  to  colonize  in  certain 
organs  and  tissues  in  preference  to  others. 

Supposing  accident  to  determine  the  tissue  in  which  the  primary  in- 
fection has  taken  place,  a  longer  or  shorter  residence  in  that  tissue, 
with  the  resulting  more  or  less  marked  acquired  immunity  against  the 
defensive  activities  of  that  tissue,  endow  the  organism  with  a  higher 
degree  of  virulence  for  it  than  for  other  tissues,  so  that  if  at  some 
future  time  the  organism  entering  the  circulation  of  a  new  host  were 
able  to  colonize  in  any  tissue  of  the  body,  its  activities  could  be 
more  easily  and  more  successfully  manifested  in  that  to  which  it  had 
already  become  accustomed,  and  to  which  it  had  acquired  a  peculiar 
adaptability.  This  adaptability  has  been  made  the  subject  of  inter- 
esting experimental  demonstration  by  Forssnerf  in  his  work  upon  the 
intravenous  injection  of  streptococci. 

SPECIAL  PHENOMENA  OF  INFECTION  AND  IMMUNITY. 

Certain  phenomena  which  present  themselves  in  the  course 
of  infection  and  immunity,  to  which  reference  has  already 
been  made,  must  now  be  considered  in  detail.  These  are 
Specific  Precipitation,  Agglutination,  Antibody  Formation, 
and  Cytolysis. 

I.  Specific  Precipitation. — In  1897  Krausi  in  studying- 
the  "specific  reactions  produced  by  homologous  serums  with 
germ-free  filtrates  of  bouillon  cultures,  of  cholera,  typhoid  and 
plague  bacteria,"  observed  that  immune  serum  brought  into 
contact  with  the  respective  culture  filtrate  occasioned  a 
precipitate  specific  in  nature,  to  which  he  gave  the  name 
"specific  precipitate."  It  thus  became  known  that  in  ad- 
dition to  antitoxins,  bacteriolysins,  and  agglutinins,  other 
specific  bodies  were  formed. 

Bordet§  and   Tchist o witch  ||  showed  that  the  phenome- 

*"  Wiener   klin.    Woch.,"    1905,  Nos.    9,    14,    16,   17;   "  Berl.  klin. 
Woch.,"  1905,  No.  15;  "Zeitschr.  f.  Hyg.,"  1905,  Bd.  I,  No.  3. 
f'Nordiskt  Medicinskt  Archiv,"  Bd.  xxxv,  p.  1,  1902. 
J  "Wiener  klin.  Woch.,"  1897,  No.  32. 
§  "Ann.  de  1'Inst.  Pasteur,'.'  1899,  p.  173. 
|]  "  Ann.  de  1'Inst.  Pasteur,"  1899,  p.  406. 


The  Specific  Precipitins 


147 


non  was  of  wide  occurrence  and  had  a  broad  significance,  for 
they  discovered  that  when  the  serum  of 
one  animal  was  injected  into  another  ani- 
mal some  reaction  took  place  by  which  the 
two  serums  being  subsequently  brought 
together  precipitation  took  place.  The 
same  was  found  true  of  milk.  Myers,* 
Jacoby,f  Nolf,|  and  others  showed  that 
the  faculty  of  provoking  specific  precipi- 
tins  was  common  to  many  albuminous 
bodies — albumen,  globulin,  albumose, 
peptone,  ricin,  etc.  Kraus  in  his  original 
communication  dwelt  upon  the  specific 
nature  of  the  precipitation,  and  was 
corroborated  by  Fish,  §  Wassermann,  || 
Morgenroth,  and  others,  and  it  was  shown 
that  the  reaction  was  sufficiently  accurate 
to  make  possible  the  differentiation  of 
human  and  goat's  milk.  The  most  im- 
portant practical  application,  however, 
came  through  Uhlenhuth**  and  Wasser- 
man,tt  who  made  use  of  it  for  the  dif- 
ferentiation of  bloods  for  forensic  pur- 
poses. 

Uhlenhuth  gave  rabbits  intraperitoneal 
injections  of  loc.c.  of  defibrinated  blood 
at  intervals  of  from  six  to  eight  days  and 
found  the  blood-serum  strongly  precipit-      complements* 
ant  after  the  fifth.     He  used  such  serum 
for  testing  the  reaction  with  the  bloods  of  oxen,  horses,  don- 
keys, pigs,  sheep,  dogs,  cats,  deer,  hares,  guinea-pigs,  rats, 
mice,  rabbits,  chickens,  geese,  turkeys,  pigeons,  and  men.  • 

The  method  of  making  the  test  is  important,  as  careless- 
ness of  detail  will  interfere  with  the  accuracy  of  the  result. 
The  blood  to  be  tested  is  diluted  about  i  :  100,  or  until 
it  has  a  feeble  red  color,  with  tap  water,  and  then  freed  from 

*"Centralbl.  f.  Bakt.,"  etc.,  1900,  Bd.  xxxm,  and  "The  Lancet," 
1900,  n,  p.  98. 

f'Archiv  fur  exper.  Path.  u.  Pharmak.,"  1900. 
t  "Ann.  de  1'Inst.  Pasteur,"  1900,  p.  297. 
§  "Courier  of  Medicine,"  St.  Louis,  Feb.,  1900. 
|| "  Verhandl.  d.  Kong.  f.  innere  Med.,"  1900,  S.  501,  Wiesbaden. 
**"  Deutsche  med.  Woch.,"  1900  and  1901. 

ft  "Samml.  klin.  Vortr.  v.  Volkman,"  Leipzig,  Verlag  von  Breitkopf 
and  Hartel,  1902. 


Fig.  28— Poly- 
ceptor  (E  h  r  1  i  c  h 
and  Marshall)  such 
as  can  be  conceived 
to  occur  in  hemo- 
lysis  and  bacterio- 
lysis where  various 
complements  are 
engaged.  a,  Re- 
ceptor of  bacterial 
cell ;  b,  cytophil 
group  of  the  ambo- 
ceptor;  c,  dominat- 
ing complement ;  d, 
subordinate  com- 
plement ;  a,  /?, 
com  plementophil 
groups  of  the  am- 
boceptor,  a  for  the 
dominating,  /?  for 
the  subordinate 


148  Immunity 

corpuscular  stroma  by  filtration  or  decantation.  Two  cubic 
centimeters  of  it  are  placed  in  a  small  test-tube,  and  further 
diluted  with  an  equal  quantity  of  physiological  salt  solution 
(if  more  water  be  added  a  precipitate  of  globulin  might 
take  place  and  spoil  the  experiment).  To  such  a  prepared 
blood  preparation,  from  six  to  eight  drops  of  the  immune 
serum  are  added.  If  the  diluted  blood  come  from  the  same 
kind  of  an  animal  as  that  used  to  immunize  the  animal 
furnishing  the  test  serum,  immediate  clouding  takes  place, 
and  a  flocculent  precipitate  forms.  The  precipitate  never 
occurs  with  any  other  blood.  For  those  who  desire  to 
acquaint  themselves  with  the  best  technic  for  the  per- 
formance of  experiments  with  the  specific  precipitates,  we 
refer  the  reader  to  the  book  of  Nuttall  mentioned  below, 
and  to  a  subsequent  paper  by  Nuttall  and  Inchley,*  in  which 
an  apparatus  for  the  accurate  quantitative  estimation  of 
the  precipitate  is  described. 

Wassermann  and  Schutzef  prepared  a  test  serum  by  in- 
jecting rabbits  with  human  blood,  and  tested  its  precipi- 
tating powers  upon  twenty-three  other  kinds  of  blood  and 
found  no  reaction  except  with  the  blood  of  a  baboon,  but 
the  reaction  in  that  case  was  not  nearly  so  marked  as  with 
human  blood. 

The  most  interesting  and  one  of  the  most  important  bio- 
logical applications  of  this  phenomenon  is  by  Nuttall,  whose 
work,  "Blood  Immunity  and  Blood  Relationship"  (Cam- 
bridge, 1904),  should  be  read  by  all  who  wish  to  study  the 
subject  for  its  scientific  interest  as  a  means  of  determining 
the  blood  relationship  of  animals,  or  its  practical  medico- 
legal  importance  in  recognizing  blood-stains.  Nuttall 
conies  to  the  following  conclusions: 

"  (i)  The  investigations  we  have  made  confirm  and  extend  the  ob- 
servations of  others  with  regard  to  the  formation  of  specific  precipitins 
in  the  blood-serum  of  animals  treated  with  various  sera.  (2)  These 
precipitins  are  specific,  although  they  may  produce  a  slight  reaction 
with  the  sera  of  allied  animals.  (3)  The  substance  in  serum  which 
brings  about  the  formation  of  a  precipitin,  as  also  the  precipitin  itself, 
are  remarkably  stable  bodies.  (4)  The  new  test  can  be  successfully 
applied  to  a  blood  which  has  been  mixed  with  those  of  several  other 
animals.  (5)  We  have  in  this  test  the  most  delicate  means  hitherto 
discovered  of  detecting  and  testing  bloods,  and  consequently  we  may 
hope  that  it  will  be  put  to  forensic  use." 

*  "Journal  of  Hygiene,"  1904,  iv,  p.  201. 

f  "Deutsche  med.  Wochenschrift,"  1900,  No.  30. 


The  Agglutinins  149 

The  injection  of  the  precipitinogenic  serum  into  animals 
results  in  the  formation  of  anti-precipitins  by  which  their 
activity  is  neutralized. 

II.  Agglutination. — This  phenomenon  was  first  observed 
by  Charrin  and  Roger*  in  the  course  of  some  experiments 
with  Bacillus  pyocyaneus.  They  found  that  the  bacilli 
when  introduced  into  serum  of  animals  infected  with  or 
immunized  against  cultures  of  Bacillus  pyocyaneus  ceased 
their  active  movements,  became  aggregated  in  clusters  and 
settled  to  the  bottom,  leaving  the  fluid  clear.  Observations 
confirming  and  enlarging  upon  the  observation  were  made 
by  Metschnikoff,  f  Issaeff  J  and  others.  Gruber  and  Durham  § 
made  an  elaborate  and  now  classic  study  of  the  subject,  first 
employing  the  term  "agglutination"  to  the  phenomenon, 
and  "agglutinins"  to  the  substances  in  the  serum  by  which 
v  it  might  be  brought  about.  They  found  that  when  cholera 
or  typhoid  bacilli  are  mixed  with  their  respective  immune 
serums,  the  organisms  lose  motility  and  become  aggregated 
in  clusters,  masses  or  "clumps."  They  further  showed 
the  reaction  to  be  specific  within  certain  limitations,  i.  e., 
typhoid  immune  serum  agglutinated  typhoid-like  bacilli 
but  no  others,  etc.,  and  they  saw  in  the  phenomenon  a 
practical  means  for  the  differentiation  of  different,  closely 
related  bacteria,  an  application  that  has,  indeed,  become  a 
useful  one. 

It  remained  for  Widal||  to  show  that  it  had  a  much  more 
important  application,  in  that  the  micro-organism  being 
known,  the  effect  produced  by  a  serum  upon  it  would  be  an 
indication  of  the  past  infection  of  the  animal  from  which 
the  serum  was  secured.  The  first  practical  application  was 
made  in  connection  with  typhoid  fever,  and  the  brilliant 
success  attending  it  had  led  to  the  test  being  known  as  the 
"Widal  reaction"  (q.v.). 

The  agglutinins  are  stable  substances  that  resist  drying 
and  can  be  kept  dry  and  active  for  years.  Widal  and  Sicard 
found  that  they  pass  with  difficulty  through  a  porcelain 
filter  and  do  not  dialyze.  They  are  precipitated  in  part  by 
15  per  cent,  of  sodium  chloride  that  throws  down  fibrinogen 

*  "Compte  rendu  de  la  Soc.  de  Biol.,"  1899,  p.  667. 

f'Ann.  de  1'Inst.  Pasteur,"  v.  1891. 

J  Ibid.,  vii,  1893. 

§  "  Munchener  med.  Woch,"  1896,  No.  9. 

||  "Societe  Medicale  des  Hopitaux,"  June  26,  1896. 


1 50  Immunity 

and  further  precipitated  with  magnesium  sulphate,  which 
throws  down  globulins.  They  therefore  think  they  are  inti- 
mately related  to  the  globulins  and  to  fibrinogen.  A  tem- 
perature of  60°  C.  diminishes  their  activity,  but  they  are  not 
destroyed  below  80°  C.  Sunlight  has  no  effect  upon  them. 

Metschnikoff  looks  upon  agglutination  as  preliminary  to 
phagocytosis  and  to  bacteriolysis,  and  thinks  it  the  effect 
of  enzymes  in  the  serum  preparing  and  clustering  the  bacteria 
to  be  taken  up  by  the  phagocytes.  Ehrlich  finds  in  the 
agglutinins  nothing  more  than  receptors  of  what  he  denomi- 
nates the  II  order,  each  of  which  possesses  a  zymophore  and 
an  agglutinophore  group.* 

Malvozf  found  that  the  addition  of  chemical  substances, 
such  as  safranin,  vesuvin,  and  corrosive  sublimate,  to  cul- 
tures of  the  typhoid  bacilli  would  cause  their  agglutination. 
Typhoid  bacilli  retained  on  the  Chamberland  filter  and 
washed  for  a  long  time,  could  no  longer  be  agglutinated,  and 
were  found  to  have  lost  their  flagella  and  to  be  without  mo- 
tion, and  led  Dineur,|  who  made  additional  experiments,  to 
conclude  that  agglutination  depended  upon  the  flagella. 
Malvoz§  found  that  bacteria  were  sometimes  agglutinated 
by  their  own  metabolic  products.  He  prepared  a  fresh 
culture  of  the  first  vaccine  of  the  anthrax  bacillus  by  thor- 
oughly distributing  it  through  £  c.c.  of  distilled  water,  and 
then  added  a  loopful  of  a  six-day-old  culture.  After  stand- 
ing for  a  few  hours  typical  agglutinations  were  observed 
under  the  microscope. 

H.  C.  Ernst  and  Robey||  think  that  flagella  have  nothing 
to  do  with  agglutination,  which  subsequent  experiment  has 
shown  to  be  correct,  as  many  non-flagellated  bacteria  can 
be  agglutinated  by  their  respective  serums. 

Bail,**  Joos,tt  Eisenberg  and  VollJt  have  shown  that  all 
of  the  agglutinins  possess  haptophore  and  agglutinophore 
groups,  either  of  which  may  be  destroyed  without  the  other. 
Thus  typhoid  agglutinative  serum  when  exposed  to  a  tem- 

*  See  Nothnagel's  "Specielle  Pathologic  und  Therapie,"  vm,  1901. 

f  "Ann.  de  1'Inst.  Pasteur,"  1897,  No.  6. 

t  "  Bull,  de  1'Acad.  de  Med.  de  Belgique,"  1898,  iv,  p.  705. 

§  "  Ann.  de  1'Inst.  Pasteur,"  Aug.  25,  1899, 

||  "  Trans.  Cong.  Amer.  Phys.  and  Surg.,"  1900,  p.  26. 
**  "  Archiv  f.  Hyg.,"  1902,  XLII,  Heft  4. 
tf  "Zeitschr.  f.  Hyg.,"  xxxvi,  1901.  p.  422. 
tt  Ibid.,  XL,  1902,  p.  155. 


The  Agglutinins  151 

perature  of  65°  C.  loses  the  agglutinophores,  and  no  longer 
clumps  the  bacteria,  though  it  retains  the  haptophores,  and 
when  brought  into  contact  with  the  bacteria  combines  with 
them,  producing  no  agglutination,  but  preventing  the  action 
of  any  other  agglutinogenic  serum. 

Buxton  and  Vaughan*  find  that  bacteria  differ  both  in 
their  agglutinogenic  powers  and  their  agglutinability,  both 
of  which  must  be  taken  into  account  in  studying  the  subject. 

Theobald  Smith  f  has  shown  that  there  are  two  kinds  of 
agglutinins,  one  of  which  acts  upon  the  bacteria  directly,  the 
other  through  the  flagella.  The  occurrence  of  these  two 
bodies  explains  some  of  the  incompatible  results  of  previous 
experiments. 

The  Technic  of  Agglutination  Tests. — The  subject  was 
at  first  investigated  with  reference  to  typhoid  fever,  and  a 
large  literature  soon  made  its  appearance.  From  typhoid 
fever  the  method  was  extended  to  one  after  another  of 
the  infectious  diseases,  in  many  of  which  the  agglutina- 
tion of  the  specific  bacteria  of  the  disease  by  the  pa- 
tient's serum  was  found  to  form  a  valuable  adjunct  in 
diagnosis.  While  this  clinical  application  was  extending, 
the  method  was  finding  added  usefulness  in  the  laboratory, 
for  it  was  at  once  evident  that  if  the  reaction  was  specific, 
as  it  was  soon  proved  to  be,  the  agglutination  of  the  bac- 
teria by  the  serum  not  only  proved  the  serum  to  come  from 
a  patient  or  animal  infected  by  a  given  bacterium,  but  proved 
as  well  that  the  given  bacterium  was  the  one  infecting.  Thus, 
the  phenomena  of  agglutination  came  to  be  a  recognized 
laboratory  method  for  the  differentiation  of  bacteria  of 
similar  species. 

The  reaction  is  one  of  the  most  accurate  known  to  us.  It 
is  so  specific  that  in  the  case  of  many  organisms  it  is  possible 
to  tell  from  what  original  source  they  may  have  come,  and 
always  to  tell  to  what  variety  they  belong  by  quantitative 
estimation.  It  is,  moreover,  a  simple  method  for  employ- 
ment in  large  laboratories  where  animals  may  always  be 
kept  on  hand  immunized  against  various  cultures,  so  that 
the  specific  serums  may  always  be  at  hand.  When  these 
animals  are  periodically  bled,  the  serums  can  be  sealed 
in  small  tubes  and  kept  an  almost  unlimited  length  of  time 
ready  for  use  when  opened  and  diluted. 

*  "Jour.  Med.  Research,"  July,  1904. 
f  Ibid.,  1904,  vol.  x,  p.  89. 


152  Immunity 

There  is  no  uniform  technic  by  which  to  apply  the  test. 
Scarcely  any  two  laboratories  employ  the  same  method, 
but  the  results  are  uniform  and  the  method  to  be  employed, 
provided  it  is  free  from  error,  is  that  found  most  convenient 
to  the  individual  operator. 

Certain  precautions  must  be  observed,  though  continued 
study  of  the  subject  and  the  employment  of  newer  methods 
have  shown  these  to  be  less  numerous. 

For  clinical  application  for  typhoid  fever  diagnosis,  com- 
mercial firms  now  prepare  small  cases  containing  the  neces- 
sary apparatus  and  "  Kicker's  solution"  of  dead  typhoid 
bacilli,  so  that  by  following  the  directions  the  diagnosis  can 
be  made  with  ease  by  one  inexpert  in  laboratory  manipu- 
lations. 

In  the  laboratory  the  agglutination  test  may  be  applied 
for  the  diagnosis  of  any  infectious  bacterial  disease,  pro- 
vided the  infecting  organism  be  at  hand,  or  for  the  recogni- 
tion of  any  micro-organism,  provided  specific  serums  are 
at  hand,  in  the  following  manner: 

If  possible,  a  culture  of  the  microorganism  grown  upon  agar-agar 
is  to  be  selected  for  the  purpose.  A  good-sized  platinum  loopful  of 
the  culture  is  taken  up  and  distributed  as  uniformly  as  possible  through- 
out a  few  cubic  centimeters  of  distilled  water.  This  is  best  done  by 
placing  the  water  in  a  test-tube  and  then  rubbing  the  culture  upon  the 
glass  just  above  the  level  of  the  fluid,  until  it  is  thoroughly  emulsified, 
permitting  it  to  enter  the  water  little  by  little  and,  finally,  washing  it 
all  down  into  the  fluid.  This  gives  a  distinctly  cloudy  fluid,  too  con- 
centrated to  use.  Of  this  one  adds  enough  to  each  of  a  series  of  watch- 
glasses  or  test-tubes,  each  containing  an  equal  volume  of  distilled 
water  (say  2  c.c.),  to  make  the  fluid  opalescent  by  reflected  light  though 
transparent  by  transmitted  light.  The  same  quantity  should  be  added 
to  each,  so  that  they  form  a  uniform  series.  The  serum  is  next  diluted 
or  otherwise  added  to  the  tubes,  so  that  they  receive  different  concen- 
trations in  a  series  from  the  blood  of  a  patient  running,  say,  i :  10,  i :  20, 
i :  30,  i :  40,  i :  50,  i :  60,  i :  80,  i :  TOO,  i :  150,  i :  200,  i :  300,  or  a  labora- 
tory series  with  artificially  prepared  serums  of  high  value,  running, 
perhaps,  i:  1000,  i:  2000,  1:5000,  i:  10,000,  1:50,000,  and  i:  100,000, 
or  many  times  higher  dilutions. 

If  watch-glasses  are  used,  they  are  stood  upon  a  black  surface, 
covered,  and  examined  in  fifteen,  thirty,  and  sixty  minutes  by  simply 
looking  at  the  dark  surface  through  the  fluid.  If  agglutination  occur, 
the  original  opalescence  gives  place  to  a  slightly  curdy  appearance, 
as  the  uniformly  suspended  bacteria  aggregate  in  clumps. 

If  test-tubes  are  employed,  they  are  best  observed  by  tilting  them 
and  looking  through  a  thin  layer  of  the  contained  fluid  at  a  dark  surface 
or  at  the  sky.  In  either  case  the  flocculent  collections  of  agglutinated 
bacteria  can  be  seen. 

The  test  can  also  be  made  and  observed  under  the  microscope  by 
the  hanging-drop  method,  but  in  working  with  such  small  quantities 
much  of  the  accuracy  of  the  technic  is  apt  to  be  lost. 

Some  knowledge  is  required  in  order  to  form  correct  deductions  from 


The  Antitoxins  153 

the  experiments.  Thus,  with  typhoid  bloods,  the  agglutination  of  the 
typhoid  bacillus  usually  occurs  within  an  hour  in  dilutions  of  i :  50, 
but  the  agglutinability  of  the  culture  employed  should  be  known  before 
the  experiment  is  undertaken. 

Similarly,  when  the  method  is  employed  for  the  differential  determi- 
nation of  bacteria,  the  value  of  the  serum  should  be  known  at  least 
approximately. 

The  agglutinins  when  injected  into  animals  effect  definite 
chemico-physiological  reactions  with  the  formation  of  anti- 
bodies inhibiting  their  own  activity. 

HI.  The  Antitoxins.— In  the  synopsis  of  immunity  ex- 
periments already  given  the  history  of  the  discovery  and 
development  of  the  anti-bodies  has  been  outlined,  together 
with  references  to  the  original  contributions  in  which  they 
were  made  public. 

In  the  section  upon  the  "Explanation  of  Immunity"  we 
have  seen  that  the  best  mode  of  accounting  for  the  occur- 
rence of  antitoxins  is  afforded  by  Ehrlich  in  the  lateral-chain 
theory.  He  regards  them  as  cell  haptophores — receptors — 
that  are  found  in  excess  of  the  requirements,  by  cells  fre- 
quently stimulated  by  the  presence  of  bacteria-products 
possessing  adapted  haptophores.  The  receptors  are  under 
normal  conditions  engaged  in  maintaining  the  proper  nutri- 
tion of  the  cell;  under  abnormal  conditions  (as  when  pre- 
empted by  the  inert  or  injurious  haptophores  of  the  bacterio- 
products)  are  obliged  to  increase  in  number  to  compensate 
for  the  damage  done  the  cell.  The  substances  by  which 
anti-body  formation  can  be  stimulated  must  bear  a  resem- 
blance to  the  normal  nutrient  substances  absorbed  by  the 
cells  in  that  they  are  provided  with  haptophore  groups 
corresponding  with  the  haptophore  groups  of  the  cells  and 
so  adapted  for  union  with  them.  Mineral  substances  and 
alkaloidal  substances  have  no  such  adaptations,  but  bacte- 
rial products,  the  toxalbumins  of  various  higher  plants, 
venoms,  enzymes,  and  other  proteid  combinations  have. 
The  possession  of  the  haptophore  groups  determines  whether 
or  not  the  cell  can  stimulate  anti-body  formation,  and  the 
ability  to  produce  anti-bodies  shows  the  existence  of  the 
haptophore  groups. 

The  attachment  of  the  haptophore  groups  to  the  cells  is 
usually  shown  by  morbid  action  of  the  cells  in  cases  where 
there  are  associated  toxophore  groups,  as  in  the  case  of  the 
bacterio-toxins,  but  may  not  be  discovered  if  there  are  no 
toxophore  groups.  The  combination  of  the  toxin-hap- 


1 54  Immunity 

tophores  with  the  cell-haptophores  can  be  demonstrated  in 
the  test-tube  by  crushing  the  cerebral  substance  of  a  rabbit, 
and  adding  tetanus  toxin.  The  toxin  becomes  fixed  by 
combination  with  the  cell  haptophores  or  receptors,  loses  its 
further  combining  powers  and  fails  to  affect  animals  into 
which  it  is  subsequently  injected.  The  increased  formation 
of  receptors  in  consequence  of  repeated  stimulation  has  been 
shown  by  the  effect  of  abrin  upon  the  conjunctiva.  If 
dropped  into  one  eye  until  the  conjunctiva  is  thoroughly 
immune  against  its  action,  the  cells  of  this  eye  develop  a 
greatly  increased  capacity  for  absorbing — i.  e.,  fixing — the 
abriri  as  compared  with  those  of  the  other  eye.  Thus  if  the 
two  conjunctival  membranes  be  dissected  out  and  a  certain 
quantity  of  abrin  triturated  with  each,  the  haptophores  of 
the  cells  of  the  immunized  membrane  fix  the  poison  so  that 
it  is  no  longer  able  deleteriously  to  affect  animals,  while 
no  such  effect  takes  place  with  the  other  membrane. 

The  ability  to  stimulate  the  formation  of  anti-bodies  is 
entirely  independent  of  any  toxic  action  and  is  entirely  the 
work  of  the  haptophores.  This  is  best  shown  in  the  fact  that 
diphtheria  toxin  that  has  been  heated  or  otherwise  manip- 
ulated until  its  toxic  action  is  lost,  still  retains  the  power 
of  combining  with  antitoxin,  or  of  producing  anti-bodies. 

The  cells  furnishing  the  haptophore  groups  or  receptors 
whose  presence  in  the  blood  gives  it  its  antitoxic  quality 
vary  in  number  or  quality  in  different  animals.  Thus,  in 
the  warm-blooded  animals  the  rapidity  with  which  tetanus 
toxin  is  anchored  to  the  cells  of  the  central  nervous  system 
seems  to  indicate  that  those  cells,  if  not  the  only  cells  in  the 
body  passing  the  adapted  receptors  by  which  it  is  anchored, 
are  the  chief  cells  by  which  it  is  absorbed.  In  the  alligator, 
however,  other  cells  seem  to  fix  the  toxin  before  it  reaches 
or  connects  with  those  of  the  nervous  system,  so  that  the 
alligator,  though  immune  against  the  action  of  the  toxin, 
is  able  to  make  antitoxin  as  well  as  susceptible  animals. 

Each  introduction  of  appropriate  anti-body  forming  sub- 
stance is  followed  by  an  outpouring  of  the  anti-body  far  in 
excess  of  what  would  neutralize  it,  so  that  after  a  systematic 
treatment  has  been  carried  out  for  some  time,  the  neutral- 
izing value  of  the  blood  may  be  a  thousand  times  what  would 
be  necessary  to  neutralize,  the  total  quantity  of  active  sub- 
stance introduced  into  the  animal. 

Each  anti-body  is  specific  in  action,  as  must  be  evident 


The  Antitoxins  155 

from  its  mode  of  formation.  Should  it  be  found,  however, 
that  several  active  bodies  possessed  haptophore  groups  of 
identical  structure,  the  anti-body  formed  by  any  of  them 
might  be  found  to  possess  common  neutralizing  powers  for  all. 

The  animal  whose  blood  contains  anti-bodies  enjoys 
immunity  from  the  active  body  by  which  they  were  formed 
only  so  long  as  they  are  present.  In  some  cases,  however, 
animals  that  have  been  long  subjected  to  the  immunization 
treatment,  and  whose  blood  contains  large  quantities  of 
free  antitoxin,  unexpectedly  become  abnormally  sensitive 
(hypersensitivity)  to  the  toxin,  and  may  die  after  receiving  a 
very  small  dose.  This  may  be  attributed  to  a  difference  in 
the  combining  activity  of  the  receptors  attached  to  the  cells, 
and  those  separated  and  free  in  the  serum.  If  the  former 
developed  a  greater  affinity  for  the  toxin  than  the  latter, 
it  would  unite  with  them  by  preference  and  intoxication 
ensue.  If  the  treatment  by  which  the  antitoxins  are  pro- 
duced is  interrupted,  they  immediately  begin  to  lessen  in 
quantity,  and  eventually  disappear.  Their  occurrence  in 
the  blood  determines  that  they  shall  be  found  in  all  the  body 
juices. 

Their  chemical  composition,  which  experiment  shows  to 
be  of  proteid  nature,  determines  that  when  practical  use 
is  to  be  made  of  them,  they  must  not  be  administered  by 
the  stomach,  as  digestion  is  usually  followed  by  their 
destruction.  In  infants,  the  proteid  digestion  being  feeble, 
antitoxins  pass  from  the  mother's  milk  to  the  sucking  off- 
spring without  digestion,  but  the  administration  of  anti- 
toxins by  this  method  at  later  periods  of  life  is  followed 
by  effects  too  uncertain  to  be  depended  upon.  For  practical 
therapeutic  purposes,  therefore,  the  administration  must 
always  be  made  hypodermically  or  intravenously. 

i.  Diphtheria  Antitoxin. — This  was  first  utilized  for  prac- 
tical therapeutic  purposes  by  Behring.*  As  usually  pre- 
pared by  the  administration  of  the  toxin,  it  is  essentially 
an  antitoxin  and  has  no  destructive  action  upon  the  diph- 
theria bacilli.  In  therapeutics  it  is  employed  to  neutralize 
or  "fix"  the  toxin  circulating  in  the  blood,  not  to  destroy 
the  bacilli,  or  to  effect  the  regeneration  of  the  tissues  injuri- 
ously acted  upon  by  the  toxin.  Martin  is  of  the  opinion 

*" Deutsche  med.  Wochenschrift,"  1890,  Nos.  49  and  50;  "Zeit- 
schrift  fur  Hygiene,"  etc.,  xn,  p.  i,  1892;  "Die  Blutserumtherapie," 
Berlin,  1902. 


156  Immunity 

that  such  purely  antitoxic  serums  are  inferior  to  those  con- 
taining other  immunity  products,  such  as  bacteriolysins, 
and  recommends  that  the  whole  culture  instead  of  the 
filtered  culture  be  used  in  the  immunization  of  the  animal. 
If  this  is  done,  the  bacteriolytic  effect  is  added  to  the  anti- 
toxic effects  of  the  serum. 

The  serum  may  be  used  to  prevent  or  to  cure  diphtheria. 

The  antitoxin  is  commercially  manufactured  at  present 
by  immunizing  horses  against  increasing  quantities  of  diph- 
theria toxin  until  the  proper  degree  of  immunity  has  been 
attained,  then  withdrawing  the  antitoxic  blood.  The  details 
are  as  follows: 

I.  The  Preparation  of  the  Toxin. — The  toxic  metabolic  products 
of  the  Bacillus  diphtheriae  are  for  the  most  part  freely  soluble,  and 
are  therefore  best  prepared  in  cultures  grown  in  fluid  media.     The 
medium  best  adapted  to  the  purpose  is  that  recommended  by  Theobald 
Smith.* 

To  make  it,  the  usual  meat  infusion  receives  the  addition  of  a 
culture  of  Bacillus  coli,  and  is  stood  in  a  warm  place  overnight.  The 
colon  bacilli  ferment  and  remove  the  muscle  and  other  sugars.  The 
infusion  is  then  made  into  bouillon,  titrated  so  that  the  reaction 
equals  +  1.1  when  tested  with  phenolphthalein.  It  then  receives  an 
addition  of  0.2  per  cent,  of  dextrose,  and  is  sterilized  in  the  autoclave. 
To  secure  the  b.est  toxic  product,  the  bacilli  at  hand  must  be  carefully 
studied  and  that  naturally  possessing  the  strongest  toxicogenic  power 
employed  for  the  cultures.  The  greatest  toxicity  seems  to  develop 
between  the  fifth  and  seventh  days.  If  the  culture  is  permitted  to 
remain  in  the  incubating  oven  beyond  this  period,  the  toxin  gradually 
is  transformed  to  toxoid  and  its  activity  declines.  The  fatal  dose  for 
a  250-300  gram  guinea-pig  should  be  about  0.001  c.c.  given  hypo- 
dermically. 

II.  The   Immunization  of  the  Animals. — All  commercial  manu- 
facturers of   diphtheria  antitoxic  serums  now   use  horses,  as  recom- 
mended by  Roux,  instead  of  the  sheep,  dogs,  and  goats  with  which 
the  earlier  investigators  worked.     The  horse  is  readily  immunized, 
gives  an  abundant  supply  of  blood  which  clots  readily  and  yields  a 
beautiful  clear  amber  serum. 

The  horse  selected  should  be  in  perfect  health,  and  should  be  tested 
with  mallein  and  tuberculin  to  avoid  obscure  glanders  and  tuberculosis. 

A  small  dose  of  the  toxic  bouillon — say  0.1  c.c.— should  be  given 
in  the  beginning,  as  one  occasionally  finds  exceptionally  susceptible 
animals  that  will  succumb  to  larger  doses.  If  a  marked  local  and 
general  reaction  follows,  it  may  be  better  to  try  another  animal.  If 
no  reaction  is  brought  about,  the  immunization  is  carried  on  as  rapidly 
as  possible.  The  toxin  is  injected  hypodermatically  into  the  tissues 
of  the  neck,  the  skin  being  thoroughly  cleaned  and  disinfected  before 
each  injection.  The  doses  are  cautiously  increased  and  may  often  be 
doubled  each  day.  If  any  unfavorable  symptoms  arise,  treatment 
must  be  interrupted  for  a  day  or  two.  The  animal  yields  good  anti- 
toxic serum  when  it  can  endure  several  doses  of  500  c.c.  of  the  strong 
toxin  mentioned  above. 

HI.  Bleeding. — When  the  withdrawal  of  a  small  quantity  of 
blood  by  a  hypodermic  needle  introduced  into  the  jugular  vein  shows 

*  "  Journal  of  Experimental  Medicine,"  May  and  July,  1899,  p.  373. 


The  Antitoxins  157 

that  the  serum  contains  a  maximum  antitoxic  strength  (300  to  1000 
units  per  cubic  centimeter),  the  horse  is  ready  to  bleed.  Some  horses 
can  be  bled  without  resistance,  but  most  of  them  require  to  be  fastened 
in  appropriate  stocks.  The  blood  is  taken  from  the  jugular  vein, 
which  is  superficial,  of  large  size,  and  easily  accessible.  The  skin  is 
carefully  shaved  over  an  area  about  9  square  inches  in  extent,  thor- 
oughly disinfected.  A  small  incision  is  made  over  the  center  of  the 
vein,  which  is  made  prominent  by  pressure  at  the  base  of  the  neck, 
and  the  point  of  a  small  sterile  trocar  being  inserted  in  the  incision 
through  the  skin,  it  is  directed  obliquely  upward  into  the  vein.  The 
blood  is  allowed  to  flow  through  a  sterile  tube  attached  to  the  cannula 
into  sterile  bottles  prepared  to  receive  it.  A  large  horse  may  furnish 
7  to  9  liters ;  small  horses  5  to  7  liters. 

IV.  Preparation  of  the  Serum. — The  blood  is  stood  away  in  a 
cool  place  until  the  clot  retracts  after  coagulation  and  the  clear  serum 
separates.     The  serum  is  then  withdrawn  under  strict  aseptic   pre- 
cautions.    It  is  variously  prepared  for  the  market.     Some  manufac- 
turers bottle  it  without  any  added  preservative;   some  add  a  crystal 
of  thymol;    some  Pasteurize  it;  some  add  carbolic    acid;  some  add 
trikresol. 

The  plain  serum  would  be  ideal,  but  the  danger  of  subsequent  con- 
tamination through  careless  treatment  makes  it  rather  better  to  have 
an  antiseptic  added.  Trikresol  is  probably  the  most  satisfactory  of 
these,  though  it  throws  down  a  precipitate  that  necessitates  the  fil- 
tration of  the  product,  and  leaves  the  serum  slightly  opalescent. 

V.  Determining  the  Potency  of  the  Serum. — The  potency  of  the 
serum  is  expressed  as  so  many  "immunizing  units."     Only  one  method 
of  testing  is  produced  at  the  present  time,  though  to  understand  it, 
it  seems  wise  to  mention  the  original  method  from  which  it  was  derived. 

(A)  Behring's  Method. — Behring's  unit  was  an  arbitrary  standard 
chosen  in  consequence  of  certain  conditions  existing  at  the  time  it 
was  devised.  It  is  difficult  to  understand  apart  from  the  circum- 
stances governing  its  creation,  but  may  be  defined  as  "Ten  times  the 
least  quantity  of  antitoxin  serum  that  will  protect  a  standard  (300  gram) 
guinea-pig  against  ten  times  the  least  certainly  fatal  dose  of  toxic  bouillon." 

The  method  of  determining  it  is  not  difficult  to  those  skilled  in 
laboratory  technic,  and  is  as  follows : 

1.  Determine  accurately  the  least  certainly  fatal  dose  of  a  sterile 
diphtheria  toxic  bouillon  for  a  standard  guinea-pig. 

2.  Determine  accurately  the  least  quantity  of  the  serum  that  will 
protect  the  guinea-pig  against  ten  times  the  above  determined  least 
fatal  dose  of  toxin. 

3.  Express  the  required  dose  of  antitoxic  serum  as  a  fraction  of  a 
cubic  centimeter  and  multiply  by  10 ;  the  result  is  one  unit. 

Example:  It  is  found  that  0.01  c.c.  of  a  toxic  bouillon  kills  at  least 
9  out  of  10  guinea-pigs,  and  is  therefore  the  least  certainly  fatal  dose. 
Guinea-pigs  receive  ten  times  this  dose  of  the  toxic  bouillon  plus 
varying  quantities  of  the  serum  to  be  tested,  measured  by  dilution — 
?ay  2oW  c-c-»  7 oW  c-c-»  JoW  c-c-  The  first  two  live-  The  fraction  ^V<y 
is  now  multiplied  by  10;  ^ylnr  X  10  =  ^  =  1  unit.  So  we  find 
that  each  cubic  centimeter  of  the  serum  contains  250  units. 

This  method  would  be  satisfactory  were  it  not  for  certain  variations 
in  the  toxic  bouillon  by  which  the  strength  is  worked  out.  Ehrlich,* 
in  an  elaborate  investigation  of  these  changes,  has  clearly  proved  that 
an  ever-changing  toxin  cannot  be  a  satisfactory  standard,  because 
it  does  not  possess  uniform  combining  affinity  for  the  antitoxin.  He 
shows  by  a  labored  scheme  that  the  toxicity  of  the  bouillon  is  no 

*  "  Klinisches  Jahrbuch,"  1897. 


158  Immunity 

index  to  its  antitoxin-combining  power,  which,  of  course,  must  be 
the  foundation  of  the  test.  The  toxin,  under  natural  conditions,  is 
changed  with  varying  rapidity  into  toxoids,  of  which  he  demonstrates 
three  groups — prototoxoids,  syntoxoids,  and  epitoxoids.  The  epi- 
toxoids  have  a  greater  antitoxin-combining  power  than  the  toxin 
itself,  yet  have  no  toxic  action  upon  the  guinea-pigs,  hence  cause 
confusion  in  the  results. 

To  secure  a  satisfactory  measure  of  the  antitoxic  strength  of  a 
serum,  it  is  therefore  more  important  to  first  determine  the  antitoxin- 
combining  power  of  the  toxin  or  toxic  bouillon  to  be  used,  than  to 
determine  its  guinea-pig  fatality,  and  this  is  what  Ehrlich  endeavors 
to  do. 

(B)  Ehrlich's  Method. — In  this  method  the  unit  is  the  same  as  in 
Behring's  method,  but  its  determination  is  arrived  at  by  a  very  im- 
portant modification  of  the  method,  by  which  the  standard  of  measure- 
ment is  a  special  antitoxin  of  known  strength,  by  which  the  antitoxin- 
combining  power  of  the  test  toxic  bouillon  is  first  determined.  Ehrlich 
began  by  determining  the  antitoxic  value  of  a  serum  as  accurately  as 
possible  by  the  old  method  and  then  used  that  serum  as  the  standard 
for  all  further  determinations.  The  serum  was  dried  in  a  vacuum,  and 
two  grams  of  the  dry  powder  were  placed  in  each  of  a  large  number 
of  small  vacuum  tubes,  connecting  with  a  small  bulb  of  phosphoric 
anhydride.  In  this  way  the  standard  powder  was  protected  from 
oxygen,  water,  and  other  injurious  agents  by  which  variations  in  its 
strength  could  be  initiated.  Periodically  one  of  these  tubes  was 
opened  and  the  contained  powder  dissolved  in  200  c.c.  of  a  mixture 
of  10  per  cent,  aqueous  solution  of  sodium  chloride  and  glycerin. 
The  subsequent  calculations  are  all  based  upon  the  strength  of  the 
antitoxin  powder.  In  Ehrlich's  first  test  serum  1  gram  of  the  dry 
powder  represented  1700  units.  Of  the  solution  mentioned,  1  c.c. 
represented  17  units;  ^  c.c.,  one  unit. 

Having  by  dilution — 1  c.c.  of  the  first  dilution  in  17  of  water — 
secured  the  standard  unit  of  antitoxin  in  a  convenient  bulk  for  the 
subsequent  manipulations,  it  is  mixed  with  varying  quantities  of  the 
toxic  bouillon  to  be  used  for  testing  the  new  serums,  until  the  least 
quantity  is  determined  that  will  cause  the  death  of  a  250-gram  guinea- 
pig  in  exactly  four  days,  when  carefully  injected  beneath  the  skin 
of  the  animal's  abdomen.  This  quantity  of  toxin  is  the  test  dose. 
If  the  toxic  bouillon  was  "normal"  in  constitution,  it  should  represent 
100  of  the  least  certainly  fatal  doses  that  formed  the  basis  of  the  old 
method  of  testing,  but  as  toxic  bouillons  contain  varying  quantities 
of  toxoids  it  may  equal  anywhere  from  fifty  to  one  hundred  and  fifty 
times  that  dose. 

The  test  dose  of  toxic  bouillon,  having  been  determined,  remains 
invariable  throughout  the  test  as  before,  the  serum  to  be  tested  for 
comparison  with  the  standard  being  modified.  The  calculation  is, 
however,  different  because  the  guinea-pig  is  receiving,  not  ten  times, 
but  more  nearly  one  hundred  times  the  least  fatal  dose,  and  the  quan- 
tity of  the  antitoxic  serum  that  preserves  life  beyond  the  fourth  day 
is  itself  the  unit. 

Example:  The  sample  of  serum  issued  as  the  standard  contains  17 
units  per  cubic  centimeter.  Serum  1  c.c.  +  water  16  c.c.  =  1  c.c. 
is  the  unit.  1  c.c.  of  the  dilution  containing  one  antitoxic  unit  is 
mixed  with  0.01,  0.025,  0.05,  0.075,  0.1  c.c.  of  the  toxic  bouillon.  All 
the  animals  receiving  less  than  0.1  c.c.  live.  A  new  series  is  started, 
and  the  guinea-pigs  all  weighing  exactly  250  grams  receive  1  unit  of 
the  antitoxin  plus  toxic  bouillon  0.08,  0.09,  0.095,  0.097,  0.1,  0.11, 
0.12,  etc.  It  is  found  that  all  receiving  more  than  0.097  die  in  four 


The  Antitoxins  159 

days,  but  that  the  animal  receiving  that  dose,  though  very  ill,  lives 
longer.  The  test  dose  may  then  be  assumed  to  be  0.1,  or  it  may 
be  calculated  more  closely  if  desired. 

To  test  the  serum  itself,  guinea-pigs  weighing  exactly  250  grams 
are  now  all  given  toxic  bouillon  0.1  c.c.  plus  varying  quantities  of  the 
serum — ^,  ^fa,  ^fa,  etc.  All  live  except  those  receiving  less  than 
T£7,  which  die  about  or  on  the  fourth  day.  The  serum  can  then  be 
assumed  to  have  400  units  per  cubic  centimeter  unless  it  be  desired 
to  test  more  closely. 

Standard  test  serums  for  making  tests  of  antitoxic  serums 
by  the  Ehrlich  method  were  first  shipped  at  small  expense 
from  the  Kaiserliches  Institut  fur  Serum-Therapie  at  Hochst- 
on-the-Main.  At  present  the  Hygienic  Laboratory  of  the 
United  States  Public  Health  and  Marine  Hospital  Service 
has  legal  control  of  the  manufacture  of  therapeutic  serums 
and  kindred  products  in  the  United  States,  issuing  licenses 
to  those  engaged  in  legitimate  manufacture,  and  furnishing 
a  standrad  test  serum,  similar  to  that  of  Ehrlich,  to  those 
entitled  to  receive  it. 

A  full  description  of  "  The  Immunity  Unit  for  Standard- 
izing Diphtheria  Antitoxin,"  by  M.  J.  Rosenau,  Director  of 
the  Hygienic  Laboratory,  can  be  found  in  Bulletin  No.  21 
of  the  U.  S.  Public  Health  and  Marine  Hospital  Service, 
Washington,  1905. 

As  the  quantity  to  be  injected  at  each  dose  diminishes 
according  to  the  number  of  units  per  cubic  centimeter  the 
serum  contains,  it  is  of  the  highest  importance  that  therapeu- 
tic serums  be  as  strong  as  possible.  Various  methods  of 
concentration  have  been  suggested.  Bujwid  *  and  H.  C. 
Ernst  f  found  that  when  an  antitoxic  serum  is  frozen  and 
then  thawed,  it  separates  into  two  layers,  the  upper  stratum 
watery,  the  lower  yellowish,  the  antitoxic  value  of  the 
yellowish  layer  being  about  three  times  that  of  the  original 
serum,  the  upper  layer  consisting  chiefly  of  water. 

The  most  satisfactory  method  of  securing  a  useful  concen- 
tration is  by  the  employment  of  the  globulin  precipitation 
as  recommended  by  Gibson, J  which  is  briefly  as  follows: 
The  diluted  citrated  plasma  is  precipitated  with  an  equal 
volume  of  saturated  ammonium  sulphate  solution  and  the 
antitoxic  proteins  separated  by  extracting  the  precipitate 

*"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Sept.  1897,  Bd.  xxn,  Nos. 
10  and  1 1,  p.  287. 

f  "Jour.  Boston  Soc.  of  Med.  Sci.,"  May,  1898,  vol.  n,  No.  8,  p. 
i37- 

t  "Jour.  Biol.  Chem.,"  i,  p.  161;  m,  p.  253. 


1 60  Immunity 

with  saturated  sodium  chloride  solution.  The  soluble 
antitoxic  proteins  are  then  reprecipitated  from  the  saturated 
sodium  chloride  solution  with  acetic  acid.  This  filtered 
precipitate  is  then  partially  dried  between  filter-papers  and 
dialyzed  in  running  water.  This  yields  a  final  product 
which  when  dried  in  vacuo  is  readily  soluble  in  salt  solution 
and  is  free  from  many  of  the  offensive  substances  in  the  horse 
serum.  Steinhardt  and  Bauzhaf*  found  that  the  therapeutic 
value  of  the  plasma  was  not  appreciably  impaired  through 
the  process  of  eliminating  the  albumins  and  other  non- 
antitoxic  proteins  by  the  salting  out  methods  employed, 
and  the  final  dialyzation  of  the  concentrated  product, 
thus  disproving  the  objection  of  Cruveilhierf  on  this  point. 

2.  Tetanus  antitoxin  was  first  prepared  by  Behring  and 
Kitasato.  J  It  can  be  employed  for  the  prevention  or  cure  of 
tetanus.  For  the  former  purpose,  hypodermic  injections  of  the 
serum  may  be  given  in  cases  with  suspicious  wounds,  or  the 
wounds  may  be  dusted  with  a  powder  made  by  pulverizing 
the  dried  serum.  For  treatment  the  serum  must  be  admin- 
istered in  frequently  repeated  large  doses  by  hypodermic  or 
intravenous  injection.  The  results  are  less  brilliant  than 
those  attained  with  diphtheria  antitoxin  because  of  the  avid- 
ity with  which  the  cells  of  the  central  nervous  system  take 
up  the  tetanus  toxin,  and  the  firmness  of  the  union  formed. 
An  analysis  of  a  great  number  of  cases  has,  however, 
shown  that  the  recoveries  following  the  free  administra- 
tion of  the  serum  exceed  the  recoveries  effected  by  other 
methods  of  treatment  by  about  40  per  cent. 

By  the  gradual  introduction  of  tetanus  toxin  Behring 
and  Kitasato§  have  been  able  to  produce  a  powerful  anti- 
toxic substance  in  the  blood  of  animals. 

The  method  of  obtaining  tetanus  antitoxic  serum  is  like 
that  employed  for  securing  diphtheria  antitoxic  serum 
(q.  v.). 

Madsen||  found  that  for  each  of  the  specific  poisons, 
tetanolysin  and  tetanospasmin,  a  specific  antitoxin  is  pro- 
duced, the  one  annulling  the  convulsive,  the  other  the 
hemolytic,  properties  of  the  toxin.  The  usual  therapeutic 
serums  contain  both  of  these. 

*  "Jour.  Infectious  Diseases,"  vol.  n,  pp.  202  and  264,  March,  1908. 

f  "Ann.  de  1'Inst.  Pasteur,"  1904,  xvm,  p.  249. 

J  "  Deutsche  med.  Wochenschrift,"  1890,  No.  49. 

§  Ibid. 

||  "Zeitschrift  fur  Hygiene,"  1899,  xxxm,  p.  239 


The  Antitoxins  161 

Different  standards  for  measuring  the  strength  of  the 
tetanus  toxin  and  different  definitions  of  the  unit  of  measure- 
ment are  given  in  different  countries,  so  that  great  confusion 
and  dissatisfaction  were  experienced  until  a  special  committee 
of  the  Society  of  American  Bacteriologists  met  in  New  York, 
Dec.  27  and  28,  1906,  and  in  collaboration  with  the  United 
States  Public  Health  and  Marine  Hospital  Service,  Hygienic 
Laboratory,  formulated  a  standard  unit  which  has  become 
the  legal  unit  of  measurment  for  the  United  States.  It  is 
thus  defined : 

"  The  immunity  unit  for  measuring  the  strength  of  tetanus 
antitoxin  shall  be  ten  times  the  least  quantity  of  antitetanic 
serum  necessary  to  save  the  life  of  a  35o-grain  guinea-pig 
for  ninety-six  hours  against  the  official  test  dose  of  a  standard 
toxin  furnished  by  the  Hygienic  Laboratory  of  the  Public 
Health  and  Marine  Hospital  Service."  The  unit  is  thus 
officially  denned,  Oct.  25,  1907,  in  Treasury  Circular,  No.  61. 

Testing  tetanus  antitoxic  serums  immediately  became 
a  matter  of  great  simplicity.  The  governmental  laboratory 
furnishes  the  ''test  toxin"  whose  strength  is  guaranteed, 
and  what  follows  is  a  simple  matter  of  dilution,  admixture 
with  the  serum  to  be  tested,  and  injection  animals  that  are 
carefully  observed  for  a  few  days. 

The  entire  subject,  historical,  theoretical,  and  practical, 
is  treated  in  Bulletin,  No.  43,  1908,  of  the  Hygienic  Labora- 
tory upon  "The  Standardization  of  Tetanus  Antitoxin," 
by  Rosenau  and  Anderson. 

3.  Antivenene  or  Anti-venomous  Serum. — This  was 
discovered  by  Phisalix  and  Bertrand*  and  made  practical 
for  therapeutic  purposes  by  Calmette.t  Calmette  found 
that  cobra  venom  contained  two  principles,  one  of  which, 
labile  in  nature  and  readily  destroyed  fey  heat,  was  destruc- 
tive in  action  upon  the  tissues  with  which  it  came  into  direct 
contact,  the  other,  stable  in  nature,  was  death-dealing 
through  its  action  upon  the  respiratory  centers.  By  heat- 
ing the  venoms  and  thus  destroying  the  irritative  principle, 
he  was  able  to  immunize  animals  against  the  other,  which 
he  looked  upon  as  the  important  element  of  the  venom. 
The  immunized  animals  furnished  an  anti-serum,  which 

*  "Compt.  rendu  de  1'Acad.  des  Sciences  de  Paris,"  Feb.  5,  1894, 
Tome  cxvm,  p.  356. 

t  "Compt.  rendu  de  la  Soc.  de  Biol.  de  Paris,"  10  Series,  Tome  i, 
p.  1 20,  Feb.  10,  1894. 
ii 


1 62  Immunity 

entirely  annulled  the  effect  of  the  toxin  (modified  venom) 
used  in  treating  them.  This  serum  was  found  to  protect 
rabbits  and  other  animals  against  both  modified  and  un- 
modified cobra  venom,  and  was  used  successfully  in  the 
treatment  of  a  number  of  human  beings  that  had  been  bitten 
by  cobras.  Calmette,  however,  erroneously  concluded  that 
because  in  most  venoms  studied  he  was  able  to  find  a 
larger  or  smaller  proportion  of  the  respiratory  poison,  it 
constituted  the  essential  element  of  the  venom  to  be  an- 
tagonized. Arguing  from  this  standpoint,  he  recommended 
his  antivenene  in  all  cases  of  snake-bite,  regardless  of  the 
variety  of  serpent.  C.  J.  Martin*  and  others  showed  that 
Calmette  was  wrong,  and  that  his  antivenene  was  useless 
in  the  treatment  of  the  bites  of  the  Australian  serpents, 
and  my  own  experiments  have  shown  it  to  be  useless 
in  the  treatment  of  the  bites  of  the  American  snakes.  In 
the  venoms  of  our  snakes — the  rattlesnake,  copper-head, 
and  moccasin — the  poison  is  essentially  locally  destructive 
in  action,  the  fatal  influence  upon  the  respiratory  centers 
being  of  secondary  importance,  fatalities  from  snake-bite 
being  rare  in  the  United  States.  Although  I  made  many 
attempts  to  immunize  horses  against  this  locally  destructive 
substance,  which  has  since  been  carefully  studied  by  Flexner 
and  Noguchi,f  I  was  unable  to  do  so.  Noguchi  {  and 
Madsen  and  Noguchi,§  however,  applied  Ehrlich's  principle 
to  the  investigation,  destroyed  the  toxophorous  group  of 
the  venom  molecules,  and  succeeded  in  producing  an  anti- 
serum  useful  in  antagonizing  the  active  principle — hem- 
orrhagin — of  the  Crotalus  venom. 

Antivenene  is  useful  in  the  treatment  of  cobra  invenoma- 
tion,  as  Calmette  has  shown  by  cases  treated  in  his  own 
laboratory.  The  serums  of  Noguchi  and  others  are  equally 
useful  in  their  respective  invenomations,  but  the  opportunity 
for  successfully  employing  antivenenes  is  very  small.  Few 
persons  are  bitten  in  places  and  at  times  where  the  remedy 
is  at  hand,  and  the  effects  of  venom  of  all  kinds  are  so  rapid 
that  immediate  treatment  is  required.  In  India  and  a  few 
other  reptile  infected  countries,  as  well  as  in  zoological 
gardens  where  venomous  serpents  are  kept,  and  in  labora- 
tories where  the  snakes  are  kept  for  experimental  purposes, 

*  "Intercolonial  Medical  Journal  of  Australia,"  1897,  n,  p.  537. 
t  "Journal  of  Experimental  Medicine,"  vi,  p.  277,  1901-1905. 
J  Ibid.,  vm,  p.  614,  1906.  §  Ibid.,  ix,  p.  18,  1907. 


The  Cytotoxins  163 

it  is  well  to  be  provided  with  a  supply  of  the  serum,  but  it 
has  no  wide  sphere  of  usefulness. 

4.  Miscellaneous  anti-bodies  of  many  kinds  have  been 
experimentally  produced, — anti-enzymes,  etc., — but  have 
no  practical  application.  A  knowledge  of  them  is,  however, 
essential  to  a  thorough  understanding  of  the  reactions  of 
immunity.  *In  the  synopsis  of  the  experiments  upon  im- 
munity reference  is  made  to  these  bodies  and  to  the  litera- 
ture bearing  upon  them. 

IV.  Cytotoxins.— The  reaction  takes  place  through  the 
combination  of  the  "amboceptor, "  " immune  body , "  "sub- 
stance sensibilisatrice,"  "fixateur,"  or  "desmon"  with  the 
"addiment,"  "complement,"  "alexin,"  or  "cytase, " 

Hemolysins. — The  phenomena  of  hemolysis  caused  by 
heterologous  serums  were  first  studied  by  Creite*  and  Lan- 
dois,f  who  studied  hemoglobinuria  following  transfusion. 
Subsequent  observations  were  made  upon  corpuscular 
agglutination  and  solution  by  venoms  by  Mitchell  and 
StewartJ  and  by  Flexner  and  Noguchi,§  and  upon  the  effects 
upon  corpuscles  of  warm-blooded  animals,  of  the  poisonous 
serum  of  certain  eels  by  Mosso||,  Camus  and  Gley,**  and 
Kossel.  ft  The  serious  consideration  of  the  subject  was, 
however,  deferred  until  Belfanti  and  Carbo ne  JJ  showed  that 
if  horses  were  injected  with  red  corpuscles  of  rabbits,  the 
serum  thereafter  obtained  from  the  horses  would  be  toxic 
for  rabbits;  Bordet  §§  had  shown  that  the  serum  of  guinea- 
pigs  injected  several  times  with  3  to  5  c.c.  of  the  defibrinated 
blood  of  rabbits  acquired  the  property  of  rapidly  dissolving 
the  red  corpuscles  of  the  rabbit  in  a  test-tube,  and  Ehrlich  and 
MorgenrothHII  had  shown  the  mechanism  of  the  hemolytic 
action.  From  this  time  on  the  literature  of  hemolysis  rapidly 
grew  and  the  subject  assumed  a  more  and  more  important 
place  in  the  domain  of  chemico-physiological  research. 

*  "Zeitschrift  f.  ration.  Med.,"  Bd.  xxxvi,  1869 — quoted  by  Nuttall 
in  his  "Blood  Immunity  and  Relationships." 

t  "Zur  Lehre  von  der  Bluttransfusion,"  Leipzig,  1875. 

t  "Transactions  of  the  College  of  Physicians  of  Philadelphia,"  1897, 
p.  105. 

§  "Journal  of  Exp.  Med.,"  1901-1905,  vi,  p.  277. 

||  "Archiv.  f.  Exp.  Path,  and  Pharmak.,"  xxv,  pp.  in  and  135. 
**  "Compt.  rendu  de  la  Soc.  de  Biol.  de  Paris,"  1898,  p.  129. 
ft  "Berliner  klin.  Wochenschrift,"  1898. 
it  "Jour,  de  la  R.  Acad.  d.  Med.  de  Torino,"  1898,  No.  8. 
§§  "Ann.  de  1'Inst.  Pasteur,"  1898,  xn,  688. 
HIl  "  Berliner  klin.  Wochenschrift,"  1899. 


1 64  Immunity 

The  technic  of  hemolytic  studies  is  comparatively  simple, 
and  it  is  intended  in  this  chapter  to  do  no  more  than  offer 
the  student  a  simple  method  of  performing  experiments 
which  he  can  modify  to  suit  his  own  purposes. 

The  Preparation  of  Blood-corpuscles. — For  the  study  of  hemo- 
lysis  and  hemo-agglutination  it  is  necesary  to  prepare,  a  5  per  cent, 
suspension  of  the  blood-corpuscles  in  an  isotonic  salt  (NaCl)  solution. 
To  do  this  the  blood  of  the  animal  is  permitted  to  flow  into  a  sterile 
tube  and  is  immediately  stirred  with  a  small  stick  or  a  platinum  wire 
until  completely  defibrinated.  Some  salt  solution  (0.85-0.9  per  cent.) 
is  then  added  and  the  mixture  shaken.  It  is  then  placed  in  a  sterile 
centrifuge  tube  and  rotated  until  the  corpuscles  are  packed  in  a  mass 
at  the  bottom.  The  supernatant  fluid  is  poured  off,  replaced  by  an 
equal  volume  of  salt  solution,  and  shaken  until  the  corpuscles  are  again 
thoroughly  distributed.  It  is  then  again  centrifugated  and  the  fluid 
again  poured  off,  after  which  95  parts  (by  volume  as  compared  with 
the  corpuscular  mass)  of  the  salt  solution  are  added  and  the  fluid  thor- 
oughly shaken  to  distribute  the  corpuscles.  This  slightly  greenish-red 
fluid  is  the  5  per  cent,  solution  of  corpuscles.  It  is,  of  course,  not  per- 
manent, and  easily  spoils  if  bacteria  enter.  It  also  gradually  deterior- 
ates through  changes  in  the  corpuscles,  so  that  it  is  not  usually  useful 
after  the  third  day,  even  when  kept  on  ice. 

The  hemolytic  substance  to  be  investigated  must  be  isotonic  with 
the  corpuscles  and  therefore  must  be  dissolved  in,  or  diluted  with,  the 
same  salt  solution  as  that  used  for  making  the  corpuscular  suspension. 
Neglect  to  observe  this  requirement  may  lead  to  error  by  diminishing 
the  tonicity  of  the  solution  and  inducing  spontaneous  or  hypotonic 
disintegration  of  the  corpuscles. 

To  secure  a  specifically  hemolytic  serum  one  injects  an  animal — 
say  a  rabbit  or  guinea-pig — with  lo-c.c.  doses  of  defibrinated  blood  of 
the  animal  for  whose  corpuscles  the  serum  is  to  be  made  hemolytic, 
the  doses  being  given  intraperitoneally  or  intravenously,  six  times,  at 
intervals  of  a  week.  The  animal  is  then  bled,  the  blood  permitted  to 
coagulate,  the  serum  separated  and  filtered,  if  necessary. 

The  contact  of  the  corpuscles  and  the  hemolytic  substance  is  best 
conducted  in  small  test-tubes  holding  about  2  c.c.  of  the  mixed  fluids. 
It  is  usually  best  to  work  with  a  constant  volume  of  the  blood-corpuscle 
suspension  and  varying  quantities  or  concentrations  of  the  hemolytic 
substances.  Two  observations  are  to  be  made,  one  after  thirty  minutes 
sojourn  in  the  thermostat  at  37°  C.,  the  other  after  twenty-four  hours 
in  the  ice-box,  both  observations  being  made  on  the  same  series  of  tubes. 
Hemolysis  is  shown  by  the  appearance  of  a  beautiful  clear  red  color 
of  the  formerly  cloudy  greenish  suspension.  One  must  notice  the  dif- 
ference between  partial  and  complete  hemolysis,  different  additions 
of  the  hemolytic  substance  being  required  for  these  results. 

Cytolysins. — It  was  soon  found  that  the  phenomena  of 
hemolysis  corresponded  to  those  by  which  many  other  cells, 
vegetable  and  animal,  were  destroyed  and  dissolved  through 
the  activity  of  immunity  products.  To  such  as  were  applic- 
able to  bacteria,  the  term  bacteriolysis  was  applied ;  to  such 
as  were  applicable  to  animal  cells,  the  term  cytolysis  was 
applied;  epitheliolysis,  endotheliolysis,  hemolysis,  spermato- 


The  Cytotoxins  365 

lysis,  etc.,  being  used  as  specific  terms.  Delezene*  first 
produced  a  leukolytic  or  leukocyte-destroying  serum  by 
treating  animals  with  the  leukocytes  of  a  heterogeneous 
species;  Metalnikoff , f  by  injecting  the  spermatozoa  of  one 
animal  into  another  of  another  species,  produced  a  sper- 
motoxic  or  spermolytic  serum;  von  DiingernJ  produced  a 
serum  capable  of  dissolving  the  ciliated  epithelium  scraped 
from  the  trachea  of  an  ox  by  injecting  the  dissociated  epi- 
thelial cells  into  an  animal;  Delezene  §  found  that  by 
injecting  an  animal  with  the  dissociated  liver  cells  of  a 
heterogeneous  animal,  a  hepatolytic  serum  could  be  pro- 
duced, and  so  grew  up  a  large  literature  upon  cytotoxins. 

The  technic  of  these  investigators  is  not  difficult.  It  is,  however, 
first  necessary  to  prepare  a  homogeneous  tissue  pulp  for  injection  into 
the  animal  that  is  to  furnish  immune  serum.  For  this  purpose  one  of 
the  best  forms  of  apparatus  is  that  of  Latapie.||  After  the  pulp  is  made, 


Fig.  29. — Latapie's  instrument  for  preparing  tissue  pulp. 

it  is  introduced  into  the  animal  in  the  same  manner  as  the  blood  for 
making  the  hemolytic  serum,  the  animal  bled  appropriately,  the  serum 
separated  and  filtered.  The  remaining  steps  do  not  differ  essentially. 
The  tissue  suspension,  having  about  the  same  concentration  as  the  5 
per  cent.  NaCl  suspensions  of  the  corpuscles,  is  used  as  the  constant 
quantity  and  the  immune  serum  used  as  the  variable  quantity.  The 
two  are  brought  into  contact  in  small  test-tubes,  kept  for  twenty-four 

*  "Compt.  rendu  de  1'Acad.  de  Sciences  de  Paris,"  1900. 
t  "Ann.  de  1'Inst.  Pasteur,"  1899. 
£  "Munchener  med.  Wochenschrift,"  1899. 

§  "Compt.  rendu  de  1'Acad.  de  Sciences  de  Paris,"  1900,  cxxx,  pp. 
938  and  1488. 

II  "Ann.  de  1'Inst.  Pasteur,"  1902,  xvi,  p.  947. 


1 66  Immunity 

hours  in  the  refrigerator,  and  the  amount  of  solution  gauged  by  the 
naked  eye  supplemented  by  microscopical  examination  of  the  tissue 
elements. 

Bacteriolysins. — The  first  observations  upon  bacterioly- 
sis was  made  in  1874  by  Traube  and  Gescheidel,*  who  found 
that  freshly  drawn  blood  was  destructive  to  bacteria.  The 
matter  was  pursued  by  numerous  subsequent  investigators 
and  was  explained  by  Buchner  as  depending  upon  alexines. 
Pfeifferf  described  the  peculiar  reaction  known  as  "Pfeiffer's 
phenomenon."  Ehrlich  and  Morgenrothf  and  Bordet§  de- 
scribed the  mechanism  of  cytolysis,  explaining  the  "Pfeiffer 
phenomenon"  and  paving  the  way  for  future  experiments. 

Direct  destruction  of  bacteria  by  blood-serum  and  body 
juices  is  rare,  and  occurs  only  when  the  serum  contains 
appropriate  quantities  of  both  factors  involved — i.  e.,  ambo- 
ceptor  and  complement.  For  the  usual  bacteriolytic 
investigations  it  is,  therefore,  necessary  to  consider  three 
factors:  i,  The  bacteria  to  be  destroyed;  2,  the  serum  fur- 
nishing the  complement;  and  3,  the  serum  furnishing  the 
immune  body. 

1.  The  bacteria  to  be  destroyed  should  be  prepared  in  the  form  of  a 
homogeneous  suspension  similar  to  that  employed  for  making  the  ag- 
glutination tests.     It  is  best  to  use  the  surface  growths  from  agar-agar, 
rubbed  between  glasses  or  ground  with  sodium  chlorid  solution,  added 
drop  by  drop,  in  a  mortar,  or  well  rubbed  upon  the  side  of  a  test-tube 
containing  the  fluid,  which  is  permitted  to  contact  with  the  mass  from 
time  to  time  by  inclining  the  tube  so  that  the  fluid  is  able  to  carry 
away  the  bacteria  as  they  are  distributed. 

If  quantitative  estimations  are  to  be  made,  the  number  of  bacteria 
in  the  suspension  must  be  known  or  at  least  a  standard  quantity  must 
be  employed,  as  the  destructive  process  is  a  chemical  one,  in  which  the 
destructive  agents  are  themselves  used  up. 

2.  The  serum  furnishing  the  complement  is  a  normal  serum-— that  is, 
the  serum  from  a  healthy  animal  that  has  undergone  no  manipulation. 

3.  The  serum  containing  the  amboceptor  or  the  immune  body  is 
obtained  from  an  animal  that  has  been  given  a  high  degree  of  immuni- 
zation against  the  bacterium  to  be  destroyed  or  dissolved.     If  it  is 
desirable,  any  complement  contained  in  this  serum  can  be  destroyed 
by  heating  for  a  short  time  to  a  point  just  short  of  coagulation. 

These  three  having  been  prepared,  an  appropriate  quantity  of  the 
bacterial  suspension  is  placed  in  a  small  test-tube,  and  an  appropriate 
quantity  of  the  normal  serum  added.  To  this  mixture  of  two  constants, 
varying  quantities  of  the  immune  serum  is  added  and  the  tube  stood 
away  for  twenty-four  hours  on  ice.  In  most  every  case  it  will  be  found 
that  the  immune  serum  contains  a  great  quantity  of  agglutinating 

*  "  Jahresb.  der  Schles.  Ges.  f.  vaterl.  Kultur,"  1874. 
f  "Deutsche  med.  Wochenschrift,"  1896,  No.  7. 
J  "Berliner  klin.  Wochenschrift,"  1899 
§  "Ann.  de  1'Inst.  Pasteur,"  xn,  1898. 


The  Cytotoxins  167 

substance,  so  that  the  bacteria  all  fall  to  the  bottom  in  a  short  time. 
This  is  independent  of  bacteriolysis.  The  bacterial  destruction  is 
gauged  by  the  disappearance  of  the  bacteria  which  have  been  dissolved, 
or  by  their  failure  to  grow  when  transplanted  to  appropriate  culture 
media,  showing  that  they  have  been  killed. 

By  making  the  bacterial  suspension  and  complementary  serum 
constant  quantities  (taking  care  that  not  too  many  bacteria  be  present), 
one  is  able  to  estimate  the  value  of  the  immune  serum.  By  using  the 
bacterial  suspension  and  a  heated  immune  serum  (containing  no  com- 
plement) as  constants  and  varying  the  addition  of  complementary 
serum,  one  can  estimate  the  respective  values  of  several  complementary 
serums.  By  using  both  serums  as  constant  factors  and  varying  the 
number  of  bacteria,  one  can  determine  the  exact  bacteriolytic  value  of 
the  mixture.  By  taking  out  and  planting  drops  from  time  to  time  the 
rapidity  of  bacteriolysis  can  be  determined,  and  by  plating  out  the  drops 
and  counting  the  colonies  one  may  arrive  at  percentages  of  destruction 
and  express  the  bacteriolytic  process  in  the  form  of  a  curve. 

A  peculiar  phenomenon  is  sometimes  observed  and  has 
been  studied  by  Neisser  and  Wechsberg,*  and  is  commonly 
called  by  their  names.  It  is  the  result  of  "deviation  of  the 
complement,"  the  condition  being  as  follows:  When  an 
animal  whose  blood-serum  is  normally  possessed  of  a  high 
degree  of  germicidal  power  is  immunized  by  repeated  injec- 
tions of  a  bacterial  endotoxin,  its  serum  when  examined  by 
the  usual  methods  fails  to  show  the  usual  increase  in  the 
specific  bactericidal  action  toward  that  particular  organism, 
though  it  retains  its  general  bacteria-destroying  power. 
If,  however,  the  serum  be  greatly  diluted,  its  action  is 
changed,  so  that  it  loses  its  general  bacteria-destroying  power 
and  develops  marked  increase  in  the  specific  destructive 
action  upon  the  particular  bacteria  used  in  the  experiment. 
Neisser  and  Wechsberg  attribute  the  peculiar  reaction  to  the 
fact  that  there  being  more  amboceptors  than  complements 
in  the  serum,  some  of  the  former  satisfy  their  combining 
affinities  by  attaching  themselves  to  the  bacteria,  some  by 
attaching  themselves  to  the  complement,  instead  of  forming 
combinations  of  all  three.  If  under  these  circumstances  the 
serum  containing  the  amboceptors  is  diluted  until  their 
number  becomes  approximately  equal  to  the  number  of  com- 
plements introduced,  any  deviation  resulting  from  inequality 
of  the  combining  affinities  becomes  improbable.  Bordet 
and  Gay,f  however,  have  performed  experiments  tending 
to  show  that  these  elements  do  not  really  unite,  thus  seem- 
ing to  controvert  the  theory  of  Neisser  and  Wechsberg,  and 

*"  Munch,  med.  Wochenschrift,"  XLVIII,  No.  13,  p.  697,  April  30, 
1901. 

t  "Ann.  de  1'Inst.  Pasteur,"  xx,  June  25,  1906,  No.  6,  pp.  267-498. 


1 68 


Immunity 


Bolton  *  has  shown  that  normal  serum  may  kill  relatively 
more  bacteria  when  diluted  than  when  undiluted. 


A1 


II* 


Iff 


Iff 


Fig.  30. — Diagram  illustrating  the  Neisser-Wechsberg  phenomenon 
of  "deviation  of  complement."  In  A1  the  three  black  units  (c)  repre- 
sent the  quantity  of  complement  necessary  for  the  dissolution  of  a  bac- 
terium, and  the  three  white  units  (/>)  the  intermediate  bodies  or  am- 
boceptors  through  which  they  may  act.  A2  shows  these  properly 
proportioned  units  properly  combined  and  anchored  to  the  bacterial 
cell  which  will  be  destroyed.  If  an  excess  of  amboceptor  units  be 
present,  as  is  suggested  in  Bl,  the  resulting  combinations  and  the  conse- 
quent results  may  vary  according  to  the  differing  combining  affinities. 
Thus,  B2  shows  an  unchanged  affinity,  i.  e.,  only  those  amboceptors 
unite  with  bacterial  cells  that  are  charged  with  complement.  C2  shows 
equal  affinity  of  the  amboceptors  for  complement  and  for  the  bacterial 
cell,  so  that  charged  or  uncharged  units  attach  themselves  to  the  cell, 
diminishing  the  complementary  action.  D2  shows  the  possible  result 
when  the  affinity  of  the  amboceptor  for  the  bacterial  cell  is  diminished 
after  charging  with  complement,  so  that  though  the  complement  and 
amboceptor  combine,  there  can  be  no  destruction  of  the  bacterium. 
Thus,  excess  of  the  amboceptor  units  may  "deviate  the  complement  " 
and  prevent  its  action. 

It  was  at  first  hoped  that  some  of  these  serums  and 
especially  the  bacteriolytic  serums  would  have  a  wide  thera- 

*  "The  Bacteriolytic  Power  of  the  Blood-serum  of  Hogs,"  Bull.  No. 
95  of  the  Bureau  of  Animal  Industry,  U.  S.  Dept.  of  Agriculture. 


The  Cytotoxins  169 

peutic  application  in  cases  in  which  non-toxicogenic  bacteria 
were  invading  the  body,  but  experiment  and  experience  have 
shown  that  the  laws  governing  their  action  greatly  limit 
their  application,  and  that  their  effects,  when  not  beneficial, 
are  bound  to  be  harmful.  The  difficulty  lies  in  the  fact  that 
when  we  manufacture  such  serums  we  prepare  only  the 
immune  body,  there  being  no  increase  of  the  complement. 


Fig.  31. — Schematic  representation  of  the  interfering  action  of  an- 
amboceptors  and  anti-complements.  A,  Anti-amboceptor  action:  c, 
Complement ;  am,  amboceptor ;  aa,  antiamboceptor  preventing  the  am- 
boceptor  from  connecting  with  the  cell.  B :  c,  Complement ;  ac,  anti- 
complement  preventing  the  complement  from  connecting  with  the  am- 
boceptor, am. 

To  introduce  this  by  itself  does  the  patient  no  good, 
because  in  most  cases  the  existing  infection  has  brought 
about  the  formation  of  as  much  or  more  ''immune  body" 
than  can  be  utilized  by  the  complement.  To  give  injec- 
tions of  active  bodies  that  cannot  be  utilized  is  shown  by 
Comus  and  Gley*  and  Kosself  to  be  followed  by  the  forma- 
tion of  anti-bodies — in  this  case  "  anti-immune  bodies" — by 
which  their  effect  is  neutralized.  Should  anti-immune  bodies 
be  found  by  this  meddlesome  medication,  the  state  of  the 
infected  animal  would  be  worse  than  before,  because  it  would 
now  be  preparing  that  which  by  neutralizing  the  combining 
affinities  of  its  own  immune  bodies,  would  prevent  them  from 
combining  with  the  elements  to  be  destroyed  and  so  activat- 
ing the  complements. 

No  satisfactory  method  of  experimentally  increasing  the 
complement  has  been  devised.  If,  as  Metschnikoff  supposes, 
the  complement  is  microcytase  derived  from  disintegrated 
leukocytes,  aseptic  suppurations  with  active  phagolysis 

*  "  Compte  rendu  de  1'Acad.  de  Sciences  de  Paris,"  Jan.  i,  1898,  126. 

f  "Berl.  klin.  Woch.,"  1898,  S.  152. 


1 70  Immunity 

should  result  in  marked  increase  of  the  complement.  As  a 
matter  of  fact,  this  does  take  place,  but  the  increase  is  so 
slight  that  the  serum  is  not  practically  valuable. 

Therapeutic  serums  whose  practical  application  is  based 
upon  their  cytolytic  activity  must,  of  necessity,  contain 
both  the  essential  factors  involved  in  cytolysis,  and  should 
contain  them  in  such  proportions  that,  regardless  of  other 
elements  in  the  blood,  they  can  exercise  their  combining  and 
dissolving  functions. 

We  are  unable  experimentally  to  accomplish  these  pre- 
requisites, therefore  are  not  in  the  position  to  accurately 
apply  bacteriolytic  serums  in  practice. 

V.  Complement  Fixation.— In  1901  Bordet,  while  in- 
vestigating the  nature  of  the  complementary  substance, 
made  a  discovery  that  has  now  become  of  great  importance, 
that  is,  the  "  Bordet-Gengou  phenomenon,"  or,  as  it  is  now 
known,  the  "  fixation  of  the  complement."  His  method  of 
procedure  was  as  follows:  Blood-corpuscles  were  sensitized 
with  appropriate  amboceptors  and  then  treated  with  freshly 
drawn  normal  serum.  Hemolysis  resulted.  If  now  he  added 
to  the  mixture  some  sensitized  blood-corpuscles  of  a  different 
species,  they  did  not  hemolyze.  Clearly,  the  complement  had 
been  used  up  in  the  first  hemolysis. 

He  next  found  that  if,  instead  of  employing  blood-corpuscles 
for  the  first  test,  he  used  sensitized  bacteria — i.  e.,  bacteria 
treated  with  an  immune  serum  containing  the  amboceptors 
appropriate  for  effecting  their  solution — the  complement  would 
similarly  be  used  up,  "  fixed,"  so  that  when  he  subsequently 
added  sensitized  red  blood-corpuscles  there  was  no  hemo- 
lysis. 

This  reaction  was  naturally  quantitative,  the  result  as  de- 
scribed depending  upon  the  fact  that  no  more  complement 
(normal  serum)  was  used  in  the  original  hemolysis  or  bac- 
teriolysis than  was  necessary,  and  so  none  left  "unfixed" 
to  effect  the  lysis  or  solution  of  the  second  factor  introduced. 

Bordet  interpreted  his  results  as  indicating  that  there  was 
only  one  complementary  or  solvent  substance,  and  though 
Khrlich  subsequently  published  what  he  looked  upon  as 
proofs  to  the  contrary,  the  opinion  of  Bordet  prevails. 

In  addition,  however,  Bordet's  experiments  have  been  of 
practical  use.  As  affording  a  means  of  quantitative  experi- 
mentation they  have  enabled  investigators  to  measure  the 
quantity  of  complement  in  normal  bloods  and  in  immunized 


Complement  Fixation  171 

bloods,  and  so  led  to  the  discovery  that  for  each  kind  of 
animal  and  for  each  individual  animal  the  complement  is 
subject  to  very  little  variation.  In  the  course  of  some  three 
years  they  were  followed  by  the  investigations  of  Neisser 
and  Sachs  upon  antigens,  and  made  to  subserve  the  useful 
purpose  of  recognizing  and  differentiating  antigenic  sub- 
stances. Thus,  when  a  certain  antibody  and  its  comple- 
ment are  combined  they  can  only  attach  themselves  to  the 
particular  specific  antigen  by  which  the  antibody  has  been 
developed.  But,  what  is  still  more  important,  they  have  led 
to  the  invention  of  methods  by  which  the  presence  of  specific 
amboceptors  may  be  determined  where  they  are  suspected, 
and  so  have  made  possible  means  of  arriving  at  a  correct 
diagnosis  in  certain  obscure  cases  of  disease  in  man. 

The  most  important  of  these  measures  is  the  Wassermann 
reaction  for  the  diagnosis  of  syphilis  (q.  v.).  By  careful 
perusal  of  the  chapter  upon  the  method  of  performing  the 
Wassermann  reaction  the  student  will  learn  the  general  de- 
tails of  the  technic,  and  can  modify  them  to  correspond  to 
the  requirements  of  other  cases  in  which  complement  fixa- 
tion is  to  be  studied. 


CHAPTER   V. 

METHODS  OF  OBSERVING   MICRO-ORGANISMS. 

IT  is  of  the  utmost  importance  to  examine  micro-organisms 
alive,  and  as  nearly  as  possible  in  their  normal  environment, 
then  to  supplement  this  examination  by  the  study  of  dead  and 
stained  specimens. 

The  study  of  the  living  organism  has  the  advantage  of 
showing  its  true  shape,  size,  grouping,  motility,  reproduc- 
tion, and  natural  history.  It  has  the  disadvantage  of  being 
somewhat  difficult  because  of  its  small  size  and  trans- 
parency. 

So  long  as  bacteria  were  observed  only  in  the  natural 
condition,  however,  it  was  impossible  to  find  them  in  the 
tissues  of  diseased  animals,  and  it  was  not  until  Weigert 
suggested  the  use  of  the  anilin  dyes  for  coloring  them  that 
their  demonstration  was  made  easy  and  their  relationship 
to  pathologic  conditions  established. 

The  beauty  and  clearness  of  stained  specimens,  and  the 
ease  with  which  they  can  be  observed,  have  led  to  some  serious 
errors  on  the  part  of  students,  who  often  fail  to  realize  the 
unnatural  condition  of  the  stained  bacteria  they  observe. 
It  only  needs  a  moment's  consideration  to  show  how  dis- 
turbed must  be  the  structure  of  an  organism  after  it  has 
been  dried,  fixed,  boiled,  or  steamed,  passed  through  several 
chemic  reagents,  dehydrated  and  impregnated  with  stains, 
etc.,  to  suggest  how  totally  unnatural  its  appearance  may 
become. 

It  is,  therefore,  necessary  to  examine  every  organism, 
under  study,  in  the  living  condition,  and  to  control  all 
the  appearances  of  the  stained  specimen  by  comparison. 

L  THE  STUDY  OF  LIVING  BACTERIA. 

The  simplest  method  of  observing  live  bacteria  is  to 
take  a  drop  of  liquid  containing  them,  place  it  upon  a  slide, 
put  on  a  cover,  and  examine. 

172 


The  Study  of  Living  Bacteria  173 

While  this  method  is  simple,  it  cannot  be  recommended, 
as  evaporation  at  the  edges  causes  currents  of  liquid  to  flow 
to  and  fro  beneath  the  cover,  carrying  the  bacteria  with 
them  and  making  it  almost  impossible  to  determine  whether 
the  organisms  under  examination  are  motile  or  not.  Should 
it  be  desirable  that  such  a  specimen  be  kept  for  a  time, 
so  much  evaporation  takes  place  that  in  the  course  of 
an  hour  or  two  it  has  changed  too  much  to  be  of  further 
use. 

The  best  way  to  examine  living  micro-organisms  is  in 
what  is  called  the  hanging  drop  (Fig.  32).  A  hollow-ground 
slide  is  used,  and  with  the  aid  of  a  small  camel's-hair  pencil 
a  ring  of  vaselin  is  drawn  on  the  slide  about,  not  in,  the 
concavity  at  its  center.  A  drop  of  the  material  to  be  ex- 
amined is  placed  in  the  center  of  a  large  clean  cover-glass 


Fig.    32. — The  "  hanging  drop  "  seen  from  above  and  in  profile. 

and  then  placed  upon  the  slide  so  that  the  drop  hangs  in, 
but  does  not  touch,  the  concavity.  The  micro-organisms 
are  thus  hermetically  sealed  in  an  air  chamber,  and  appear 
under  almost  the  same  conditions  as  in  the  culture.  Such  a 
specimen  may  be  kept  and  examined  from  day  to  day,  the 
bacteria  continuing  to  live  until  the  oxygen  or  nutriment 
is  exhausted.  By  means  of  a  special  apparatus  (Fig.  33), 
in  which  the  microscope  is  placed,  the  growing  bacteria  may 
be  watched  at  any  temperature,  and  very  exact  observa 
tions  made. 

The  hanging  drop  should  always  be  examined  at  the 
edge,  as  the  center  is  too  thick. 

In  such  a  specimen  it  is  possible  to  determine  the  shape, 
size,  grouping,  division,  sporulation,  and  motility  of  the 
organism  under  observation. 


174       Methods  of  Observing  Micro-organisms 

Care  should  be  exercised  to  use  a  rather  small  drop, 
especially  for  the  detection  of  motility,  as  a  large  one  vi- 
brates and  masks  the  motility  of  the  sluggish  forms. 

When  the  bacteria  to  be  observed  are  in  solid  or  semi- 
solid  culture,  a  small  quantity  of  the  culture  should  be  mixed 
in  a  drop  of  sterile  bouillon  or  other  fluid.' 

For  observing  the  growth  of  bacteria  where  it  is  desirable 
to  prevent  movement,  Hill  *  has  invented  an  ingenious 
device  which  he  calls  the  "  hanging  block.''  His  directions 
for  preparing  it  are  as  follows:  + 

"Pour  melted  nutrient  agar  into  a  Petri  dish  to  the  depth  of  about 
one-eighth  or  one-quarter  inch.  Cool  this  agar,  and  cut  from  it  a 
block  about  one-quarter  inch  to  one-third  inch  square  and  of  the  thick- 
ness of  the  agar  layer  in  the  dish.  This  block  has  a  smooth  upper  and 
under  surface.  Place  it,  under  side  down,  on  a  slide  and  protect  it 
from  dust.  Prepare  an  emulsion,  in  sterile  water,  of  the  organism 
to  be  examined  if  it  has  been  grown  on  a  solid  medium,  or  use  a  broth 
culture;  spread  the  emulsion  or  broth  upon  the  upper  surface  of  the 
block  as  if  making  an  ordinary  cover-slip  preparation.  Place  the 
slide  and  block  in  a  37°  C.  incub'ator  for  five  to  ten  minutes  to  dry 
slightly.  Then  lay  a  clean  sterile  cover-slip  on  the  inoculated  surface 
of  the  block  in  close  contact  with  it,  usually  avoiding  air-bubbles. 
Remove  the  slide  from  the  lower  surface  of  the  block  and  invert  the 
cover-slip  so  that  the  agar  block  is  uppermost.  With  a  platinum 
loop  run  a  drop  or  two  of  melted  agar  along  each  side  of  the  agar 
block,  to  fill  the  angles  between  the  sides  of  the  block  and  the  cover- 
slip.  This  seal  hardens  at  once,  preventing  slipping  of  the  block. 
Place  the  preparation  in  the  incubator  again  for  five  or  ten  minutes 
to  dry  the  agar-agar  seal.  Invert  this  preparation  over  a  moist  cham- 
ber and  seal  the  cover-slip  in  place  with  white  wax  or  paraffin.  Vaselin 
softens  too  readily  at  37°  C.,  allowing  shifting  of  the  cover-slip.  The 
preparation  may  then  be  examined  at  leisure." 

With  this  means  of  examining  the  growing  cultures,  Hill 
has  acquired  interesting  knowledge  of  the  fission  and  budding 
of  Bacillus  diphtherias 

If  the  specimens  to  be  examined  must  be  kept  for  some 
time  at  an  elevated  temperature,  some  such  apparatus  as 
that  of  Nuttall  will  be  found  useful  though  expensive. 


II.  STAINING  BACTERIA. 

In  the  early  days  of  bacteriology  efforts  were  made  to  facili- 
tate the  observation  of  bacteria  by  the  use  of  nuclear  dyes. 
Both  carmin  and  hematoxylin  tinge  the  nuclei  of  the  bac- 
teria a  little,  but  so  unsatisfactorily  that  since  Weigert  in- 
troduced the  anilin  dyes  for  the  purpose,  all  other  stains 

*  "Journal  of  Medical  Research,"  vol.  vn,  No.  2;  new  series,  vol.  n, 
March,  1902. 


Staining  Bacteria 


175 


'have  been  abandoned.  The  affinity  between  the  bacteria 
and  the  anilin  dyes  is  peculiar,  and  in  certain  cases  can  be 
used  for  the  differentiation  of  species. 

The  best  anilin  dyes  made  at  the  present  time,  and  those 
which  have  become 
the  standard  for  all 
bacteriologic  work, 
are  made  in  Ger- 
many by  Dr.  Grii- 
bler,  and  in  or- 
dering stains  the 
name  of  this  man- 
ufacturer should  be 
specified. 

Readers  interest- 
ed in  the  biochemis- 
try of  the  subject 
will  do  well  to  refer 
to  the  excellent 
papers  by  Arnold 
Grimme,*  upon 
"The  Important 
Methods  of  Stain- 
ing Bacteria,  etc.," 
and  Marx,f  upon 
"  The  Metachro- 
matic  and  Babes- 
Ernst  Granules." 

In  this  work 
special  methods  for 
staining  such  bac- 
teria as  have  pecu- 
liar reactions  will 
be  given  together 
with  the  descrip- 
tion of  the  particular  organisms,  general  methods 
being  discussed  in  this  chapter. 

Preparations  for  General  Examination. — For  bacterio- 
logic purposes  thin  covers  (No.  i)  are  required,  because 
thicker  glasses  may  interfere  with  the  focussing  of  the  oil- 
immersion  lenses.  The  cover-glasses  must  be  perfectly 

*  "Centralbl.  f.  Bakt.,"  etc.,  Bd.  xxxn,  Nos.  2,  3,  4,  and  5,  1902. 

f  Ibid.,  xxxii,  Nos.  10  and  u,  p.  108,  1902. 


Fig.  33. — Apparatus  for  keeping  objects 
under  microscopic  examination  at  constant 
temperatures  (Nuttall). 


only 


176       Methods  of  Observing  Micro-organisms 

dean.  It  is  therefore  best  to  clean  a  large  quantity  in  ad- 
vance of  use  by  immersing  them  first  in  a  strong  mineral 
acid,  then  washing  them  in  water,  then  in  alcohol,  then  in 
ether,  and  finally  keeping  them  in  ether  until  they  are  to 
be  used.  Except  that  it  sometimes  cracks,  bends,  or 
fuses  the  edge  of  the  glass,  a  more  convenient  method  is 
to  wipe  the  glasses  as  clean  as  possible  with  a  soft  cotton 
cloth,  seize  them  with  fine-pointed  forceps,  and  pass  them 
repeatedly  through  a  small  Bunsen  flame  until  it  becomes 
greenish-yellow.  The  hot  glass  must  then  be  slowly  elevated 
above  the  flame,  so  as  to  allow  it  to  anneal.  This  manceuver 
removes  the  organic  matter  by  combustion.  It  is  not 
expedient  to  use  covers  twice  for  bacteriologic  work,  though 
if  well  cleansed  by  immersion  in  acid  and  washing,  they 
may  subsequently  be  employed  for  ordinary  microscopic 
objects. 

The  fragility  of  the  covers  and  their  likelihood  to  be 
broken  or  dropped  at  the  critical  moment,  make  most 
workers  prefer  to  stain  directly  upon  the  slide.  The  slide 
should  be  thoroughly  cleaned,  and  if  the  material  to  be 
examined  is  spread  near  one  end,  the  other  may  serve  as  a 
convenient  handle.  The  slide  is  also  to  be  preferred  if 
a  number  of  examinations  are  to  be  made  simultaneously 
or  for  comparison,  as  it  is  large  enough  to  contain  a  number 
of  ''smears." 

Simple  Method  of  Staining. — The  material  to  be  ex- 
amined must  be  spread  in  the  thinnest  possible  layer  upon 
the  surface  of  the  perfectly  clean  cover-glass  or  slide  and  dried. 
The  most  convenient  method  of  spreading  is  to  place  a 
minute  drop  on  the  glass  with  a  platinum  loop,  and  then 
spread  it  evenly  over  the  glass  with  the  flat  wire.  Should 
it  be  stained  at  once  it  would  all  wash  off,  so  it  must  next 
be  fixed  to  the  glass  by  being  passed  three  times  through  a 
flame,  experience  having  shown  that  when  drawn  through 
the  flame  three  times  the  desired  effect  is  usually  accom- 
plished. The  Germans  recommend  that  a  Bunsen  burner 
or  a  large  alcohol  lamp  be  used,  that  the  arm  describe  a 
circle  a  foot  in  diameter,  each  revolution  occupying  a  second 
of  time,  and  the  glass  being  made  to  pass  through  the  flame 
from  apex  to  base  three  times.  This  is  supposed  to  be  exactly 
the  requisite  amount  of  heating.  The  rule  is  a  good  one  for 
the  inexperienced. 

Inequality  in  the  size  of  various  flames  may  make  it  de- 


Simple  Method  of  Staining  177 

sirable  to  have  a  more  accurate  rule.  Novy*  suggests  that 
as  soon  as  it  is  found  that  the  glass  is  so  hot  that  it  can  no 
longer  be  held  against  the  finger  it  is  sufficiently  heated  for 
fixing. 

After  fixing,  the  preparation  is  ready  for  the  stain.  Every 
laboratory  should  be  provided  with  "  stock  solutions,"  which 
are  saturated  solutions  of  the  ordinary  dyes.  For  pre- 
paring them  Wood*  gives  the  following  parts  per  100  as 
being  sufficiently  accurate: 

Alcoholic  solutions  (96  per  cent,  alcohol).  Aqueous  solutions  (distilled  water). 

Fuchsin. .3.0  grams. 

Gentian  violet 4.8  Gentian  violet 1.5  grams. 

Methylene-blue 7.0  Methylene-blue    .....  .6.7 

(70  per  cent,  alcohol). 

Scharlach  R 3.2      " 

Soudan  III 0.2      " 

(50  per  cent,  alcohol). 
Thionin .  .  ..0.6      "  Thionin.  .  ..1.2      " 


Of  these  it  is  well  to  have  fuchsin,  gentian  violet,  and  meth- 
ylene-blue  always  made  up.  The  stock  solutions  will  not 
stain,  but  form  the  basis  of  the  staining  solutions.  For 
ordinary  staining  an  aqueous  solution  is  employed.  A  small 
bottle  is  nearly  filled  with  distilled  water,  and  the  stock 
solution  added,  drop  by  drop,  until  the  color  becomes  just 
sufficiently  intense  to  prevent  the  ready  recognition  of  ob- 
jects through  it.  For  exact  work  it  is  probably  best  to  give 
these  stains  a  standard  composition,  using  5  c.c  of  the 
saturated  alcoholic  solution  to  95  c.c.  of  water.  Such  a 
watery  solution  possesses  the  power  of  readily  penetrating 
the  dried  cytoplasm  of  the  bacterium. 

Cover-glasses  are  apt  to  slip  from  the  fingers  and  spill  the 
stain,  so  when  using  them  it  is  well  to  be  provided  with 
special  forceps  (Fig.  34),  which  hold  the  glass  in  a  firm  grip 
and  allow  of  all  manipulations  without  danger  of  soiling 
the  fingers  or  clothes.  The  ordinary  sharp-pointed  forceps 
are  unfit  for  the  purpose,  as  capillary  attraction  draws 
the  stain  between  the  blades  and  makes  certain  the  soiling 
of  the  fingers.  In  using  the  special  forceps  the  glass  should 
not  be  caught  at  the  edge,  but  a  short  distance  from  it,  as 

*  "Laboratory  Work  in  Bacteriology,"  1899. 

t  "  Chemical  and  Microscopical  Diagnosis,"  N.  Y.,  1905,  D.  Apple- 
ton  &  Co.,  p.  683 


178       Methods  of  Observing  Micro-organisms 

shown  in  the  cut.  This  altogether  prevents  capillary  at- 
traction between  the  blades.  When  the  material  is  spread 
upon  the  slide  no  forceps  are  needed,  and  the  method  corre- 
spondingly simplified.  Sufficient  stain  is  allowed  to  run 
from  a  pipet  upon  the  smear  to  flood  it,  but  not  overflow, 
and  is  allowed  to  remain  for  a  moment  or  two,  after  which  it 
is  thoroughly  washed  off  with  water.  The  smear  upon  a 
slide  is  then  dried  and  examined  at  once,  a  drop  of  oil  of 
cedar  being  placed  directly  upon  the  smear,  and  no  cover- 
glass  used.  If  the  staining  has  been  done  upon  a  cover-glass, 
it  can  be  mounted  upon  a  slide  with  a  drop  of  water  between, 
and  then  examined,  though  this  is  less  satisfactory  than  ex- 
amination after  mounting  in  Canada  balsam. 


Fig.  34. — Stewart's  cover-glass  forceps 

Sometimes  the  material  to  be  examined  is  solid  or  too 
thick  to  spread  upon  the  glass  conveniently.  Under  such 
circumstances  a  drop  of  distilled  water  or  bouillon  can  be 
added  and  a  minute  portion  of  the  material  mixed  in  it  and 
spread  upon  the  glass. 

When  the  bacteria  are  contained  in  urine  or  other  non- 
albuminous  fluid,  so  that  the  heat  used  for  fixing  has  nothing 
to  coagulate  and  fix  the  organisms  to  the  glass,  a  drop  of 
Meyer's  glycerin-albumen  can  be  added  with  advantage, 
though  the  precaution  must  be  taken  to  see  that  this  mix- 
ture contains  no  bacteria  to  cause  confusion  with  those 
in  the  material  to  be  studied. 

The  entire  process  is,  in  brief:  (i)  Spread  the  material 
upon  the  glass;  (2)  dry — do  not  heat;  (3)  pass  three 
times  through  the  flame;  (4)  stain — one  minute;  (5)  wash 
thoroughly  in  water;  (6)  dry;  (7)  mount  in  Canada 
balsam. 

To  Observe  Bacteria  in  Sections  of  Tissue. — Har- 
dening.— It  not  infrequently  happens  that  the  bacteria  to 
be  examined  are  scattered  among  or  inclosed  in  the  cells 
of  tissues.  Their  demonstration  then  becomes  a  matter 


Staining  Bacteria  in  Tissues  179 

of  difficulty,  and  the  method  employed  must  be  modified 
according  to  the  particular  kind  of  organism.  The  success 
of  the  method  will  depend  upon  the  good  preservation  of 
the  tissue  to  be  studied.  As  bacteria  disintegrate  rapidly 
in  dead  tissue,  the  specimen  for  examination  should  be 
secured  as  fresh  as  possible,  cut  into  small  fragments,  and 
immersed  in  absolute  alcohol  from  six  to  twenty-four  hours, 
to  kill  and  fix  the  cells  and  bacteria.  The  blocks  are  then 
removed  from  the  absolute  alcohol  and  kept  in  80  to  90  per 
cent,  alcohol,  which  does  not  shrink  the  tissue.  Solutions 
of  bichlorid  of  mercury*  may  also  be  used  and  are  par- 
ticularly useful  when  the  bacteria  are  to  be  studied  in 
relation  to  the  cells  of  the  tissues. 

Tissues  preserved  in  95  per  cent,  alcohol,  Muller's  fluid, 
4  per  cent,  formaldehyd,  and  other  ordinary  solutions 
rarely  show  the  bacteria  well. 

Embedding. — The  ordinary  methods  of  embedding  suffice. 
The  simpler  of  these  are  as  follows : 

/.  Celloidin  (Schering). — The  solutions  of  celloidin  are 
made  in  equal  parts  of  absolute  alcohol  and  ether  and 
should  have  the  thickness  of  oil  or  molasses.  From  the 
hardening  reagent  (if  other  than  absolute  alcohol)  pass  the 
blocks  of  tissue  through: 

Ninety-five  per  cent,  alcohol,  twelve  to  twenty-four 
hours ; 

Absolute  alcohol,  six  to  twelve  hours; 

Thin  celloidin  (consistence  of  oil),  twelve  to  twenty- 
four  hours; 

Thick  celloidin  (consistence  of  molasses),  six  to  twelve 
hours. 

Place  upon  a  block  of  vulcanite  or  hard  paraffin,  allow  the 
ether  to  evaporate  until  the  block  can  be  overturned  without 
dislodging  the  specimen;  then  place  in  80  per  cent,  alcohol 

*  Zenker's  fluid: 

Bichromate  of  potassium 2.5  grams 

Sulphate  of  sodium 1.0  gram 

Bichlorid  of  mercury . .  .  < 5.0  grams 

Water 100.0       " 

At  the  time  of  using  add  5  grams  of  glacial  acetic  acid.  Permit 
the  specimens  to  remain  in  the  solution  for  a  few  hours  only,  then  wash 
for  twenty-four  hours  in  running  water  and  transfer  to  80  per  cent, 
alcohol. 


180      Methods  of  Observing  Micro-organisms 

until  ready  to  cut.     The  knife  must  be  kept  flooded  with 
alcohol  while  cutting. 

Celloidin  is  soluble  in  absolute  alcohol,  ether,  and  oil  of 
cloves,  so  that  the  staining  of  the  sections  must  be  accom- 
plished without  the  use  of  these  reagents  if  possible. 

Celloidin  sections  can  be  fastened  to  the  slide,  if  desired, 
by  firmly  pressing  filter  paper  upon  them  and  rubbing  hard, 
then  allowing  a  little  vapor  of  ether  to  run  upon  them. 

//.  Paraffin. — Pure  paraffin  having  a  melting-point  of 
about  55°  C.  is  used.  The  hardened  blocks  of  tissue  are 
passed  through: 

Ninety-five  per  cent,   alcohol,   twelve  to   twenty-four 

hours ; 

Absolute  alcohol,  six  to  twelve  hours; 
Chloroform,  benzole,  or  xylol,  four  hours; 
A  saturated  solution  of  paraffin  in  one  of  the  above 
reagents,  four  to  eight  hours. 

The  block  is  then  placed  in  melted  paraffin  in  an  oven 
or  paraffin  water-bath,  at  5o°-6o°  C.,  until  the  volatile 
reagent  is  all  evaporated,  and  the  tissue  impregnated  with 
paraffin  (four  to  twelve  hours),  and  finally  embedded  in 
freshly  melted  paraffin  in  any  convenient  mold.  In  cutting, 
the  knife  must  be  perfectly  dry. 

The  cut  paraffin  sections  can  be  placed  upon  the  surface 
of  slightly  warmed  water  to  flatten  out  the  wrinkles,  and 
then  floated  upon  a  clean  slide  upon  which  a  film  of  Meyer's 
glycerin-albumen  (equal  parts  of  glycerin  and  white  of  egg 
thoroughly  beaten  up  and  filtered,  and  preserved  with  a 
crystal  of  thymol)  has  been  spread.  After  drying,  the  slides 
are  placed  in  the  paraffin  oven  for  an  hour  at  60°  C.,  so 
that  the  albumen  coagulates  and  fixes  the  sections  to  the 
glass. 

When  sections  so  spread  and  fixed  upon  the  slide 
are  to  be  stained,  the  paraffin  must  first  be  dissolved  in 
chloroform,  benzole,  xylol,  oil  of  turpentine,  etc.,  which 
in  turn  must  be  removed  with  95  per  cent,  alcohol.  The 
further  staining,  by  whatever  method  desired,  is  accom- 
plished by  dropping  the  reagents  upon  the  slide. 

///.  Glycerin-gelatin. — As  the  penetration  of  the  tissue 
by  celloidin  is  attended  with  deterioration  in  the  staining 
qualities  of  the  tubercle  bacillus,  it  has  been  recommended 


Staining 


181 


by  Kolle  *  that  the  tissue  be  saturated  with  a  mixture  of 
glycerin,  i  part ;  gelatin,  2  parts ;  and  water,  3  parts ;  cemented 
to  a  cork  or  block  of  wood,  hardened  in  absolute  alcohol, 
and  cut  as  usual  for  celloidin  with  a  knife  wet  with  alcohol. 

Staining. — Simple  Method. — For  ordinary  work  the 
following  simple  method  can  be  recommended:  After  the 
sections  are  cut  and  cemented  to  the  slide,  the  paraffin 
and  celloidin  should  be  removed  by  appropriate  solvents. 
The  sections  are  immersed  in  the  ordinary  aqueous  solution 
of  the  anilin  stain  and  allowed  to  remain  about  five  minutes, 
next  washed  in  water  for  several  minutes,  then  decolorized 
in  0.5  to  i  per  cent,  acetic  acid  solution.  The  acid  removes  the 
stain  from  the  tissues,  but  ultimately  from  the  bacteria  as 
well,  so  that  one  must  watch  carefully,  and  so  soon  as  the 
color  has  almost  disappeared  from  the  sections,  they  must 
be  removed  and  transferred  to  absolute  alcohol.  At  this 
point  the  process  may  be  interrupted  to  allow  the  tissue 
elements  to  be  countercolored  with  alum-carmin  or  any 
stain  not  requiring  acid  for  differentiation,  after  which  the 
sections  are  dehydrated  in  absolute  alcohol,  cleared  in 
xylol,  and  mounted  in  Canada  balsam. 

The  greater  number  of  applications  can  be  made  by 
simply  dropping  the  reagents  upon  the  slide  while  held  in 
the  fingers.  Where  expos- 
ure to  the  reagents  is  to  be 
prolonged,  the  Coplin  jar 
(Fig-  35)  or  some  more 
capacious  device  must  be 
employed. 

Pfeiffer's  Method.— The 
sections  are  stained  for  one- 
half  hour  in  diluted  Ziehl's 
carbol-fuchsin  (pp.  187  and 
716),  then  transferred  to 
absolute  alcohol  made  feeb- 
ly acid  with  acetic  acid. 
The  sections  must  be  care- 

fully  watched,  and  so  soon  35._Coplin's  staining  jar. 

as     the     original,     almost 

black-red  color  gives  place  to  a  red-violet  color  they  are 
removed  to  xylol,  to  be  cleared  preparatory  to  mounting 
in  balsam. 

*  Flugge's  "  Die  Mikroorganismen."  vol.  i,  page  534. 


CROSS-SECTION 
SHOWING  SLIDES 
M  POSITION, 


1 82       Methods  of  Observing  Micro-organisms 

Loffler's  Method. — Certain  bacteria  that  do  not  permit 
ready  penetration  by  the  dye  require  some  more  intense 
stain.  One  of  the  best  of  these  is  Loffler's  alkaline  methy- 
lene-blue : 

Saturated  alcoholic  solution  of  methylene-blue .  .    30 
1  :  10,000  aqueous  solution  of  caustic  potash  .  .  100 

The  cut  sections  of  tissue  are  stained  for  a  few  minutes 
and  then  differentiated  in  a  i  per  cent,  solution  of  hydro- 
chloric acid  for  a  few  seconds,  after  which  they  are  dehy- 
drated in  alcohol,  cleared  in  xylol,  and  mounted  in  balsam. 

Bacteria,  such  as  the  typhoid  fever  bacillus,  which  de- 
colorize rapidly,  do  not  require  the  use  of  acid  for  the 
differentiation,  washing  in  water  or  alcohol  being  sufficient. 

Gram's  Method  of  Staining  Bacteria  in  Tissue. — Gram 
was  the  fortunate  discoverer  of  a  method  of  impregnating 
bacteria  with  an  insoluble  color.  It  will  be  seen  at  a 
glance  that  this  is  a  marked  improvement  on  the  methods 
given  above,  as  the  stained  tissue  can  be  washed  thoroughly 
in  either  water  or  alcohol  until  its  cells  are  colorless,  without 
fear  that  the  bacteria  will  be  decolorized.  The  details  of 
the  method  are  as  follows:  The  section  is  stained  from  five 
to  ten  minutes  in  a  solution  of  a  basic  anilin  dye,  pure  anilin 
(anilin  oil)  and  water.  This  solution,  first  devised  by 
Bhrlich,  is  known  as  Bhrlich's  solution.  The  ordinary 
method  of  preparing  it  is  to  mix  the  following: 

Pure  anilin : 4 

Saturated  alcoholic  solution  of  gentian  violet.  .    11 
Water 100 

Instead  of  gentian  violet,  methyl  violet,  Victoria  blue,  or 
any  pararosanilin  dye  will  answer.  The  rosanilin  dyes, 
such  as  fuchsin,  methylene-blue,  vesuvin,  etc.,  will  not  react 
with  iodin,  and  so  cannot  be  used  for  the  purpose.  The 
anilin-oil  solutions  do  not  keep  well;  in  fact,  seldom  longer 
than  six  to  eight  weeks,  sometimes  not  more  than  two  or 
three;  therefore  it  is  best  to  prepare  but  a  small  quantity 
by  pouring  about  i  c.c.  of  pure  anilin  into  a  test-tube, 
filling  the  tube  about  one-half  with  distilled  water,  shaking 
well,  then  filtering  as  much  as  is  desired  into  a  small  dish. 
To  this  the  saturated  alcoholic  solution  of  the  dye  is  added 
until  the  surface  becomes  distinctly  metallic  in  appear- 
ance. 


Staining  183 

Friedlander  recommends  that  the  section  remain  from 
fifteen  to  thirty  minutes  in  warm  stain,  and  in  many  cases 
the  prolonged  process  gives  better  results. 

From  the  stain  the  section  is  given  a  rather  hasty  washing 
in  water,  and  then  immersed  from  two  to  three  minutes  in 
Gram's  solution  (a  dilute  Lugol's  solution) : 

lodin  crystals 1 

Potassium  iodid 2 

Water ,300 

The  specimen  while  in  the  Gram  solution  turns  a  dark 
blackish-brown  color,  but  when  removed  and  carefully 
washed  in  95  per  cent,  alcohol  again  becomes  blue.  The 
washing  in  95  per  cent,  alcohol  is  continued  until  no  more 
color  is  given  off  and  the  tissue  assumes  its  original  color. 
If  it  is  simply  desired  to  find  the  bacteria,  the  section  can 
be  dehydrated  in  absolute  alcohol  for  a  moment,  cleared 
in  xylol,  and  mounted  in  Canada  balsam.  If  it  is  necessary 
to  study  the  relation  of  the  bacteria  to  the  tissue  elements, 
a  nuclear  stain,  such  as  alum-carmin  or  Bismarck  brown, 
may  be  previously  or  subsequently  used.  Should  a  nuclear 
stain  requiring  acid  for  its  differentiation  be  desirable,  the 
process  of  staining  must  precede  the  Gram  stain,  so  that 
the  acid  shall  not  act  upon  the  stained  bacteria. 

Gram's  method  rests  upon  the  fact  that  the  combination 
of  mycoprotein,  anilin  dye,  and  the  iodids  forms  a  compound 
insoluble  in  alcohol. 

The  process  described  may  be  summed  up  as  follows: 

Stain  in  Ehrlich's  anilin- water  gentian  violet  five  to 

thirty  minutes ; 
Wash  in  water; 

Immerse  two  to  three  minutes  in  Gram's  solution; 
Wash  in  95  per  cent,  alcohol  until  no  more  color  comes 

out; 

Dehydrate  in  absolute  alcohol; 
Clear  in  xylol; 
Mount  in  Canada  balsam. 

This  method  stains  the  majority  of  bacteria,  but  not  all, 
hence  can  be  used  to  aid  in  the  differentiation  of  similar 
species: 


184       Methods  of  Observing  Micro-organisms 

Gram-negative.  Gram -positive. 

Bacillus  anthracis  symptomatici ;        Bacillus  aerogenes  capsulatus; 
Bacillus  coli  (whole  group);  Bacillus  anthracis; 

Bacillus  ducreyi;  Bacillus  botulinus; 

Bacillus  dysenteriae;  Bacillus  diphtherias; 

Bacillus  icteroides;  Bacillus  subtilis  (whole  group); 

Bacillus  influenzae;  Bacillus  tetani; 

Bacillus  mallei;  Bacillus  tuberculosis  (whole  acid- 

Bacillus  cedematis  maligni;  fast  group); 

Bacillus  pestis  bubonica;  Diplococcus  pneumonias; 

Bacillus  pneumonias  (Friedlander) ;     Micrococcus  tetragenus; 
Bacillus  proteus  vulgaris;  Staphylococcus  pyogenes  albus; 

Bacillus  pyocyaneus;  Staphylococcus  pyogenes  aureus; 

Bacillus  rhinoscleromatis;  Streptococcus  pyogenes. 

Bacillus  suipestifer; 
Bacillus  suisepticus; 
Bacillus  typhosus  (whole  group) ; 
Diplococcus  intracellularis  meningitidis; 
M  icrococcus  cat  arrh  al  is ; 
Micrococcus  gonorrhreae  (Neisser) ; 
Micrococcus  melitensis; 
Spirillum  cholerae  asiaticae; 
Spirillum  cholerae  gallinarum; 
Spirillum  cholerae  nostras; 
Spirillum  metschnikovi; 
Spirillum  tyrogenum; 
Spirochaete  duttoni; 
Spirochaete  obermeieri; 
Spirochaete  refringens; 
Treponema  pallidum; 
Treponema  pertenue. 

No  matter  how  carefully  the  method  is  performed,  an 
unsightly  precipitate  is  sometimes  deposited  upon  the 
tissue,  obscuring  both  its  cells  and  contained  bacteria. 
Muir  and  Ritchie  obviate  this  (i)  by  making  the  staining 
solution  with  i  :  20  aqueous  solution  of  carbolic  acid  instead 
of  the  saturated  anilin  solution,  and  (2)  by  clearing  the 
tissue  with  oil  of  cloves  after  dehydration  with  alcohol. 
The  oil  of  cloves,  however,  is  itself  a  powerful  decolorant 
and  must  be  washed  out  in  xylol  before  the  section  is 
mounted  in  Canada  balsam. 

Gram's  method  is  also  employed  to  aid  in  differentiat- 
ing similar  species  of  bacteria  in  culture.  A  thin  layer  of 
a  suspension  of  the  bacteria  to  be  examined  is  spread  upon 
a  slide  or  cover-glass,  dried,  and  fixed;  then  flooded  with 
the  anilin-oil  gentian  violet  or  other  staining  solution. 
The  solution  is  kept  warm  by  holding  the  glass  flooded 
with  the  stain  over  a  small  flame.  The  process  of  staining 
is  continued  from  two  to  five  minutes.  If  the  heating 
causes  the  stain  to  evaporate,  more  of  it  must  be  added 
so  that  it  does  not  dry  and  incrust  the  glass. 


Staining  185 

The  stain  is  poured  off,  and  replaced  by  Gram's  solution, 
which  is  allowed  to  remain  from  one-half  to  two  minutes, 
and  gently  agitated. 

The  smear  is  next  washed  in  95  per  cent,  alcohol  until 
the  blue  color  is  wholly  or  almost  lost,  after  which  it  can 
be  counterstained  with  eosin,  Bismarck  brown,  vesuvin, 
etc.,  washed,  dried,  and  mounted  in  Canada  balsam.  Given 
briefly,  the  method  is : 

Stain  with  Ehrlich's  solution  two  to  five  minutes; 

Gram's  solution  for  one-half  to  two  minutes; 

Wash  in  95  per  cent,  alcohol  until  decolorized; 

Counterstain  if  desired;  wash  off  the  counterstain  with  water; 

Dry; 

Mount  in  Canada  balsam. 

Nicolle*  suggests  the  following  modification  of  the  technic : 

a.  For  Cover-glass  Specimens: 

1 .  Stain  for  one  to  five  minutes  in  a  warm  solution  made  as  fol- 

lows: 10  c.c.  of  saturated  alcoholic  solution  of  gentian  violet, 
100  c.c.  of  a  i  per  cent,  aqueous  solution  of  carbolic  acid. 

2.  Immerse  from  four  to  six  seconds  in  the  iodine-iodide  of  potas- 

sium solution. 

3.  Decolorize  in  a  mixture  of  3  parts  of  absolute  alcohol  and  i 

part  of  acetone. 

4.  Counter  stain  if  desired. 

b.  For  Sections: 

1.  Stain  the  nuclear  elements  of  the  tissue  with  carmine.     For 

this  Nicolle  prefers  Orth's  carmine  solution  (5  parts  of  Orth's 
carmine  with  i  part  of  95  per  cent,  alcohol). 

2.  Stain  in  the  carbol-gentian  violet,  as  indicated  above. 

3.  Immerse  for  four  to  six  seconds  in  the  iodine-iodide  of  potas- 

sium solution. 

4.  Differentiate  with  absolute  alcohol  containing  0.33  per  cent. 

(by  volume)  of  acetone. 

5.  Treat  with  95  per  cent,  alcohol  containing  some  picric  acid 

until  the  tissue  is  greenish  yellow  (one  to  five  seconds). 

6.  Dehydrate  with  absolute  alcohol. 

7.  Clear  with  xylol  or  other  appropriate  reagent. 

8.  Mount  in  balsam. 

The  Gram-Weigert  Stain  can  be  employed  with  beautiful 
results  for  staining  many  micro-organisms.  It  differs  from 
the  Gram  method  in  that  anilin  oil  instead  of  alcohol  is 
used  for  decolorizing.  To  secure  the  most  brilliant  results 
it  is  best  first  to  stain  the  tissue  with  alum,  borax,  or 
lithium  carmin,  and  then — 

*  "Ann.  del'Inst.  Pasteur,"  1895,  ix. 


1 86       Methods  of  Observing  Micro-organisms 

1.  Stain   in    Ehrlich's   anilin-oil-water   gentian   violet, 

five  to  twenty  minutes; 

2.  Wash  off  excess  with  normal  salt  solution; 

3.  Immerse  in  dilute  iodin  solution  (iodin  i,  iodid  of 

potassium  2,  water  100)  for  one  minute; 

4.  Drain  off  the  fluid  and  blot  the  section  spread  out 

upon  the  slide,  with  absorbent  paper; 

5.  Decolorize  with  a  mixture  of  equal  parts  of  anilin 

and  xylol; 

6.  Wash  out  the  anilin  with  pure  xylol; 

7.  Mount  in  xylol  balsam. 

Eosin  and  Methylene-blue  (Mallory)  make  a  beautiful 
contrast  tissue  stain  for  routine  work,  and  also  demonstrates 
the  presence  of  most  bacteria.  The  success  of  the  method 
seems  to  depend  largely  upon  the  quality  of  the  reagents 
used  and  a  careful  study  of  their  effects.  Hardening  in 
Zenker's  fluid  is  highly  recommended  as  a  preliminary.  The 
details  as  given  by  Mallory  are  as  follows: 

1.  Stain  paraffin  sections  in  a  5  to  10  per  cent,  aqueous 

solution  of  eosin  for  five  to  twenty  minutes  or 
longer; 

2.  Wash  in  water  to  get  rid  of  the  excess  of  eosin; 

3.  Stain   in   Unna's   alkaline   methylene-blue   solution 

(methylene-blue  i,  carbonate  of  potassium  i,  water 
100),  diluted  i  :  10  with  water,  for  one-half  to  one 
hour,  or  use  a  stronger  solution  and  stain  for  a  few 
minutes  only; 

4.  Wash  in  water. 

5.  Differentiate  and  dehydrate  in  95  per  cent,  alcohol, 

followed  by  absolute  alcohol  until  the  pink  color 
returns  in  the  section; 

6.  Clear  with  xylol; 

7.  Mount  in  xylol  balsam. 

The  nuclei  and  micro-organisms  will  be  colored  blue,  the 
cytoplasm,  etc.,  red. 

Zieler*  recommends  for  the  staining  of  the  typhoid, 
glanders  and  other  difficultly  stainable  bacteria,  the  follow- 
ing method  of  demonstration  in  the  tissues  :- 

*  "Centralbl.  f.  allg.  Path.  u.  path!  Anat."  Bd.  xiv,  No.  14,  p.  561. 


Staining  187 

1.  Fix  and  harden  in  Miiller-formol  solution. 
Paraffin  imbedding. 

2.  Staining  overnight  in  Orcein  D.  (Griibler),   0.1 

Officinal  "schwefelsaiire"  (sulphuric  acid), 2.0 

70  per  cent,  alcohol, lOo'o 

3.  Washing  in  70  per  cent,  alcohol  for  a  short  time  to  remove 

the  excess  of  orcein. 

4.  Washing  in  water. 

5.  Staining  in  polychrome  methylene-blue  ten  minutes  to  two 

hours. 

6.  Washing  in  distilled  water. 

7.  Thorough  differentiation  in  glycerin -ether  1 :  2-5  water  until 

the  tissues  become  pale  blue. 

8.  Washing  in  distilled  water. 

9.  Seventy  per  cent,  alcohol. 

10.  Absolute  alcohol. 

11.  Xylol. 

12.  Balsam. 

Glanders  bacilli  appear  dark  violet  on  a  colorless  back- 
ground; typhoid  bacilli  intense  dark  red  violet. 

Method  of  Staining  Spores. — It  has  already  been  pointed 
out  that  the  peculiar  quality  of  the  spore  capsules  pro- 
tects them  to  a  certain  extent  from  the  influence  of  stains 
and  disinfectants.  On  this  account  they  are  much  more 
difficult  to  color  than  the  adult  bacteria.  Several  methods 
are  recommended,  the  one  generally  employed  being  as 
follows :  Spread  the  thinnest  possible  layer  of  material  upon 
a  cover-glass,  dry,  and  fix.  Have  ready  a  watch-crystalful 
of  Ehrlich's  solution,  preferably  made  of  fuchsin,  and  drop 
the  cover-glass,  prepared  side  down,  upon  the  surface,  where 
it  should  float.  Heat  the  stain  until  it  begins  to  steam, 
and  allow  the  specimen  to  remain  in  the  hot  stain  for  from 
five  to  fifteen  minutes.  The  cover  is  then  transferred  to  a 
3  per  cent,  solution  of  hydrochloric  acid  in  absolute  alcohol 
for  about  one  minute.  Abbott  recommends  that  the  cover- 
glass  be  submerged,  prepared  side  up,  in  a  dish  of  this  solu- 
tion and  gently  agitated  for  exactly  one  minute,  removed, 
washed  in  water,  and  counterstained  with  an  aqueous  solu- 
tion of  methyl  or  methylene-blue. 

In  such  a  specimen  the  spores  should  appear  red,  and  the 
adult  organisms  blue. 

I  have  not  found  that  spores  usually  color  so  easily, 
and  for  many  species  the  best  method  seems  to  be  to  place 
the  prepared  cover-glass  in  a  test-tube  half  full  of  carbol- 
f  uchsin : 

Fuchsin ! 

Alcohol    10 

Five  per  cent  aqueous  solution  of  phenol  crys- 
tals .  100 


1 88       Methods  of  Observing  Micro-organisms 

and  boil  it  for  at  least  fifteen  minutes,  after  which  it  is 
decolorized,  either  with  3  per  cent,  hydrochloric  or  2-5  per 
cent,  acetic  acid,  washed  in  water,  and  counterstained  blue. 

Muir  and  Ritchie  *  recommend  that  cover-films  be  pre- 
pared and  stained  as  for  tubercle  bacilli  (q.  V.),  decolor- 
ized with  a  i  per  cent,  sulphuric  acid  solution  in  water  or 
methyl  alcohol,  then  washed  in  water  and  counterstained 
with  a  saturated  aqueous  methylene-blue  solution  for  half  a 
minute,  washed  again  with  water,  dried,  and  mounted  in 
Canada  balsam. 

Abbott's  method  of  staining  spores  is  as  follows: 

1.  Stain  deeply  with  methylene-blue,  heating  repeatedly  until  the 

stain  reaches  the  boiling-point — one  minute. 

2.  Wash  in  water. 

3.  Wash  in  95  per  cent,  alcohol  containing  0.2  to  0.3  per  cent,  of 

hydrochloric  acid. 

4.  Wash  in  water. 

5.  Stain  for  eight  to  ten  seconds  in  anilin-fuchsin  solution. 

6.  Wash  in  water. 
7-  Dry. 

8.  Mount  in  balsam. 

The  spores  are  blue;  the  bacteria,  red. 

Mollerf  finds  it  advantageous  to  prepare  the  films,  before 
staining,  by  immersion  in  chloroform  for  two  minutes,  fol- 
lowing this  by  immersion  in  5  per  cent,  chromic  acid  solu- 
tion for  one-half  to  two  minutes. 

The  exact  technic  is  as  follows: 

1.  Treat  the  spread  with  chlororoform  for  two  minutes. 

2.  Wash  with  water. 

3.  Treat  with  5  per  cent,  solution  of  chromic  acid  for  one-half  to 

two  minutes. 

4.  Wash  in  water. 

5.  Stain  with  carbol-fuchsin,  slowly  heating  until  the  fluid  boils. 

6.  Decolorize  in  5  per  cent,  aqueous  sulphuric  acid. 

7.  Wash  well  with  water. 

8.  Stain  in  a  i :  100  aqueous  solution  of  methylene-blue  for  thirty 

seconds. 
The  spores  should  be  red  and  the  bacilli  blue. 

Anjeszky  {  recommends  the  following  method  of  staining 
spores,  which  is  said  always  to  give  good  results  even  with 
anthrax  bacilli:  A  cover-glass  is  thinly  spread  with  the 
spore-containing  fluid  and  dried.  While  it  is  drying,  some 
0.5  per  cent,  hydrochloric  acid  is  warmed  in  a  porcelain 

*  "Manual  of  Bacteriology,"  London,  1897. 

f  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Bd.  x,  p.  273. 

i  Ibid.,  Feb.  27,  1898,  xxm,  No.  8,  p.  329. 


Staining  189 

dish  over  a  Bunsen  flame  until  it  steams  well  and  bubbles 
begin  to  form.  When  the  solution  is  hot  and  the  smear 
dry,  the  cover-glass  is  dropped  upon  the  fluid,  which  is 
allowed  to  act  upon  the  unfixed  smear  for  three  or  four 
minutes.  The  cover  is  removed,  washed  with  water,  dried, 
and  fixed  for  the  first  time,  then  stained  with  Ziehl's  carbol- 
fuchsin  solution,  which  is  warmed  twice  until  fumes  arise. 
The  preparation  is  allowed  to  cool,  decolorized  with  a  4-5 
per  cent,  sulphuric  acid  solution,  and  counterstained  for  a 
minute  or  two  with  malachite  green  or  methylene-blue. 
The  whole  procedure  should  not  take  longer  than  eight  to 
ten  minutes. 

Fiocca*  suggests  the  following  rapid  method:  "About 
20  c.c.  of  a  10  per  cent,  aqueous  solution  of  ammonium  are 
poured  into  a  watch-glass,  and  10  to  20  drops  of  a  saturated 
solution  of  gentian  violet,  fuchsin,  methyl  blue,  or  safranin 
added.  The  solution  is  warmed  until  vapor  begins  to  rise, 
then  is  ready  for  use.  A  very  thinly  spread  cover-glass, 
carefully  dried  and  fixed,  is  immersed  for  three  to  five 
minutes  (sometimes  ten  to  twenty  minutes),  washed  in 
water,  washed  momentarily  in  a  20  per  cent,  solution  of 
nitric  or  sulphuric  acid,  washed  again  in  water,  then  counter- 
stained  with  an  aqueous  solution  of  vesuvin,  chrysoidin, 
methyl  blue,  malachite  green,  or  safranin,  according  to  the 
color  of  the  preceding  stain.  This  whole  process  is  said  to 
take  only  from  eight  to  ten  minutes,  and  to  give  remarkably 
clear  and  beautiful  pictures." 

Method  of  Staining  Flagella. — This  is  somewhat  more 
difficult  than  the  staining  of  the  bacteria  or  their  spores. 

Loffler's  Method.} — This  is  the  original  and  best  method, 
though  somewhat  cumbersome,  and  hence  rarely  employed 
at  the  present  time.  Three  solutions  are  used : 

(A)- 

Twenty  per  cent,  aqueous  solution  of  tannic 
acid3..  10 

Cold  saturated  aqueous  solution  of  ferrous  sul- 
phate   5 

Alcoholic  solution  of  fuchsin  or  methyl  violet 

(B)  One  per  cent,  aqueous  solution  of  caustic  soda. 

(C)  An  aqueous  solution  of  sulphuric  acid  of  such  strength 
that    i   c.c.   will  exactly  neutralize    an   equal  quantity  of 
solution  B. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  July  i,  1893,  xiv,  No.  i. 
f  Ibid.,  1890,  Bd.  vii,  p.  625. 


i  go       Methods  of  Observing  Micro-organisms 

Some  of  the  culture  to  be  stained  is  mixed  upon  a  cover- 
glass  with  a  drop  of  distilled  water  making  a  first  dilution, 
which  is  still  too  rich  in  bacteria  to  permit  the  flagella  to 
show  well,  so  that  it  is  recommended  to  prepare  a  second 
by  placing  a  small  drop  of  distilled  water,  upon  a  cover 
and  taking  a  loopful  from  the  first  dilution  to  make  the 
second,  and  spreading  it  over  the  entire  surface  without 
much  rubbing  or  stirring.  The  film  is  allowed  to  dry,  and 
is  then  fixed  by  passing  it  three  times  through  the  flame. 
When  this  is  done  with  forceps  there  is  some  danger  of  the 
preparation  becoming  too  hot,  so  Loffler  recommends  that 
the  glass  be  held  in  the  fingers  while  the  passes  through  the 
flame  are  made. 

The  cover-glass  is  now  held  in  forceps,  and  the  mordant, 
solution  A,  dropped  upon  it  until  it  is  well  covered,  when 
it  is  warmed  until  it  begins  to  steam.  The  mordant  must 
be  replaced  as  it  evaporates.  It  must  not  be  heated  too 
strongly;  above  all  things,  must 'not  boil.  This  solution 
is  allowed  to  act  from  one-half  to  one  minute,  is  then  washed 
off  with  distilled  water,  and  then  with  absolute  alcohol 
until  all  traces  of  the  solution  have  been  removed.  The 
real  stain, — Loftier  recommends  an  anilin- water  fuchsin 
(Ehrlich's  solution), — which  should  have  a  neutral  reaction, 
is  next  dropped  on  so  as  to  qover  the  film,  and  heated  for 
a  minute  until  vapor  begins  to  arise,  after  which  it  is  washed 
off  carefully,  dried,  and  mounted  in  Canada  balsam.  To 
obtain  the  neutral  reaction  of  the  stain,  enough  of  the  i 
per  cent,  sodium  hydrate  solution  is  added  to  an  amount 
of  the  anilin-water-fuchsin  solution  having  a  thickness  of 
several  centimeters  to  begin  to  change  the  transparent 
into  an  opaque  solution. 

A  specimen  thus  treated  may  or  may  not  show  the  flagella. 
If  not,  before  proceeding  further  it  is  necessary  to  study 
the  chemic  products  of  the  micro-organism  in  culture 
media.  If  by  its  growth  the  organism  elaborates  alkalies, 
from  i  drop  to  i  c.c.  of  solution  C  in  16  c.c.  must  be  added 
to  the  mordant  A,  and  the  staining  repeated.  It  may  be 
necessary  to  stain  again  and  again  until  the  proper  amount 
is  determined  by  the  successful  demonstration  of  the  fla- 
gella. On  the  other  hand,  if  the  organism  by  its  growth 
produces  acid,  solution  B  must  be  added,  drop  by  drop, 
and  numerous  stained  specimens  examined  to  see  with 
what  addition  of  alkali  the  flagella  will  appear.  Loffler 


Staining  191 

fortunately  worked  out  the  amounts  required  for  some 
species,  and  of  the  more  important  ones  the  following 
solutions  of  B  and  C  must  be  added  to  16  c.c.  of  solution 
A  to  attain  the  desired  effect: 

Cholera  spirillum £-1   drop  of  solution  C  •$ 

Typhoid  fever 1  c.c.  of  solution  B 

Bacillus  subtilis 28-30  drops  of  solution  B 

Bacillus  of  malignant  edema  .36  or  37  drops  of  solution  B 

Part  of  the  success  of  the  staining  depends  upon  having 
the  bacteria  thinly  spread  upon  the  glass,  and  as  free  from 
albuminous  and  gelatinous  materials  as  possible.  The 
cover-glass  must  be  cleaned  most  painstakingly;  too  much 
heating  in  fixing  must  be  avoided.  After  using  and  washing 
off  the  mordant,  the  preparation  should  be  dried  before  the 
application  of  the  anilin-water-fuchsin  solution. 

Pitfield's  Method. — Pitfield*  has  devised  a  single  solution,  at 
once  mordant  and  stain.  It  is  made  in  two  parts,  which  are 
filtered  and  mixed: 

(A)- 

Saturated  aqueous  solution  of  alum 10  c.c. 

Saturated  alcoholic  solution  of  gentian  violet  .  .    1    " 

(B)- 

Tannic  acid     " 1  gram 

Distilled  water  10  c.c. 

The  solutions  should  be  made  with  cold  water,  and  im- 
mediately after  mixing  the  stain  is  ready  for  use.  The 
cover-slip  is  carefully  cleaned,  the  grease  being  burned  off 
in  a  flame.  After  it  has  cooled,  the  bacteria  are  spread 
upon  it,  well  diluted  with  water.  After  drying  thoroughly 
in  the  air,  the  stain  is  gradually  poured  on  and  by  gentle 
heating  brought  almost  to  a  boil;  the  slip  covered  with  the 
hot  stain  is  laid  aside  for  a  minute,  then  washed  in  water 
and  mounted. 

Smith's  Modification  of  Pitfield' s  Method.* — A  boiling  saturated 
solution  of  bichlorid  of  mercury  is  poured  into  a  bottle  in  which 
crystals  of  alum  have  been  placed  in  quantity  more  than  sufficient 
to  saturate  the  fluid.  The  bottle  is  shaken  and  allowed  to  cool; 
10  c.c.  of  this  solution  are  added  to  the  same  volume  of  freshly 
prepared  tannic  acid  solution  and  5  c.c.  of  carbol  fuchsin  added. 
Mix  and  filter.  The  filtrate,  which  is  the  mordant,  is  caught 

*  "Medical  News,"  Sept.  7,  1895. 

t  ''British  Medical  Journal,"  1901,  i,  p.  205. 


192       Methods  of  Observing  Micro-organisms 

directly  upon  the  spread  (the  liquid  must  always  be  filtered  at  the 
time  of  use)  and  heated  gently  for  three  minutes,  but  not  permitted 
to  boil.  Wash  with  water  and  then  stain  in  the  following: 

Saturated  alcoholic  solution  of  gentian  violet i  c.c. 

Saturated  solution  of  ammonium  alum 10 

Filter  the  stain  also  directly  upon  the  slide  at  the  time  of 
using,  and  heat  it  for  three  to  four  minutes.  Wash  thoroughly 
in  water,  dry,  and  mount  in  balsam. 

Van  Ermengem's  Method. — Van  Ermengem  *  has  devised 
a  somewhat  complicated  method  of  staining  flagella,  which 
has  given  great  satisfaction.  Three  solutions,  which  he 
describes  as  the  bain  fixateur,  bain  sensibilisateur,  and  bain 
reducteur  et  reinforcateur,  are  to  be  used  as  follows: 

1.  Bain  fixateur: 

2  per  cent,  solution  of  osmic  acid    1  part 

10-25  per  cent,  solution  of  tannin 2  parts 

The  cover-glasses,  which  are  very  thinly  spread,  dried, 
and  fixed,  are  placed  in  this  bath  for  one  hour  at  the  room 
temperature,  warmed  until  steam  arises,  and  then  kept  hot 
for  five  minutes.  They  are  next  washed  with  distilled 
water,  then  with  absolute  alcohol,  then  again  with  distilled 
water.  All  three  washings  must  be  very  thorough. 

2.  Rain  sensibilisateur: 

5  per  cent,  solution  of  nitrate  of  silver  in  distilled  water. 

The  films  are  allowed  to  remain  in  this  for  a  few  seconds, 
and  are  then  immediately  transferred  to  the  third  bath. 

3.  Bain  reducteur  et  reinforcateur: 

Gallic  acid 5  grams 

Tannin 3       " 

Fused  potassium  acetate    10       " 

Distilled  water 350  c.c. 

The  preparations  are  kept  in  this  solution  for  a  few 
seconds,  then  returned  to  the  nitrate  of  silver  solution  until 
they  begin  to  turn  black.  They  are  then  washed,  dried, 
and  mounted. 

Mervyn  Gorden  modifies  the  method  by  allowing  the 
preparations  to  remain  in  the  second  bath  for  two  minutes, 
transferring  to  the  third  bath  for  one  and  a  half  to  two 

*  "Travaux  du  Lab.  d'hygiene  et  des  bact.  de  Gand.,"  t.  I,  p.  3. 
Abstracted  in  the  "  Central  bl.  f.  Bakt.  u.  Parasitenk.,"  1894,  Bd.  xv, 
p.  969. 


Staining  193 

minutes,  and  then  washing,  drying,  and  mounting  without 
returning  to  the  second  bath. 

Muir  and  Ritchie  find  it  advantageous  to  use  a  fresh 
supply  of  the  third  solution  for  each  specimen. 

Rossi*  gives  the  following  directions  for  staining  flagella : 

The  culture  to  be  examined  should  be  a  young  culture,  not  more 
than  ten,  eighteen,  or  twenty-four  hours  old.  It  should  be  made  upon 
freshly  prepared  agar-agar,  or  upon  the  reagent  after  it  has  been  melted 
and  then  congealed,  as  it  is  of  the  utmost  importance  that  the  surface 
be  moist.  The  culture  should  be  examined  by  the  hanging-drop  method 
to  see  that  the  organisms  are  actively  motile  before  the  staining  is  at- 
tempted. 

The  staining  should  be  done  only  after  the  greatest  care  has  been 
taken  to  see  that  all  the  conditions  are  favorable.  For  this  reason  the 
cover-glasses  employed  in  making  the  spreads  must  be  carefully  cleaned 
with  alcohol,  then  immersed  in  steaming  sulphuric  acid  for  ten  to  fifteen 
minutes.  They  are  then  washed  in  water,  then  placed  in  a  mixture  of 
alcohol  and  benzine  (equal  parts),  wiped  with  a  clean  soft  cloth,  and 
passed  through  the  colorless  Bunsen  flame  forty  to  fifty  times,  and 
then  that  side  of  the  glass  utilized  for  the  "spread"  that  has  been  in 
direct  contact  with  the  flame. 

A  platinum  loopful  of  the  appropriate  culture  is  placed  in  a  drop  of 
distilled  water  upon  a  clean  slide  and  slightly  stirred.  If  conditions 
are  favorable,  it  forms  a  homogeneous  emulsion.  If  clumps  appear, 
the  cultural  conditions  are  not  favorable. 

If  favorable,  a  loopful  of  this  dilution  is  added  to  i  c.c.  of  distilled 
water  in  a  clean  cover-glass  and  thoroughly  stirred.  From  the  center 
of  the  surface  of  this  fluid  a  platinum  loopful  is  next  taken  and  placed 
upon  each  of  the  prepared  cover-glasses  and,  without  spreading  or  stir- 
ring, allowed  to  dry  in  the  air  or  in  an  exsiccator. 

The  staining  solutions  are  made  as  follows: 

(A)  A  solution  of  50  grams  of  pure  crystalline  carbolic  acid  in 

looo  c.c.  of  distilled  water,  to  which  40  grams  of  pure  tannin 
are  added,  the  whole  being  warmed  on  a  water-bath  until 
solution  is  complete. 

(B)  Basic  fuchsin  (rosanilinchlorhydrate) 2.5  grams 

Absolute  alcohol 100.0  c.c. 

(C)  Potassium  hydrate i  .o  gram 

Distilled  water 100.0  grams 

Mix  solutions  A  and  B  and  preserve  in  a  well-closed  bottle.  Place 
solution  C  in  a  bottle  with  a  pipette  stopper.  When  the  staining  is  to 
be  done,  one  pours  15  to  20  c.c.  of  the  A  B  mixture  into  a  glass-stop- 
pered test-tube  and  adds  2  or-  3  drops  of  solution  C.  A  precipitate 
forms,  but  quickly  dissolves  on  shaking.  More  of  solution  C  is  added, 
and  the  tube  shaken  until  the  solution  becomes  brown  and  clouded  and 
one  can  see  a  fine  precipitate  in  a  thin  layer  of  the  fluid.  The  fluid  is 
next  filtered  several  times  through  the  same  filter  and  caught  in  the 
same  glass  until  it  will  remain  clear  for  several  minutes.  Then  it  is 
poured  on  the  filter  a  last  time  and  4  or  5  drops  allowed  to  fall  upon  each 
of  the  prepared  cover-glasses.  In  a  short  time  a  sheen  is  observed  upon 
the  surface  of  the  fluid  on  the  cover-glasses,  showing  that  a  fine  precipi- 
tate has  formed.  When  this  has  occurred,  a  little  experience  will  show 
when  the  proper  moment  arrives  to  throw  off  the  fluid  and  wash  the 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  xxxm,  Orig.,  1903,  p.  572. 
13 


194       Methods  of  Observing  Micro-organisms 

cover  in  distilled  water.  It  is  the  precipitate  that  clings  to  the  flagella 
and  renders  them  distinctly  visible.  If  no  precipitate  occurs,  the 
flagella  will  not  be  seen. 

L.  Smith  *  offers  the  following  modification  of  Newman's 
method  f  as  being  a  simple  and  excellent  method  of  staining 
flagella: 

The  material  and  cover-glasses  are  prepared  with  care  as 
for  the  foregoing  methods,  after  which  one  proceeds  as  follows : 

1.  Transfer  a  loopful  of  the    bacillary  emulsion  to  the  clean  slide 

or  cover-glass  and  allow  it  to  dry  in  the  air. 

2.  Expose  to  a  mild  degree  of  heat,  holding  the  glass  in  the  fingers — 

this  is  rather  drying  than  actual  heating. 

3.  Allow  the  stain  to  drop  from  a  filter  upon  the  film  and  remain 

in  contact  five  to  ten  minutes. 
The  formula  for  the  stain  is 

I.  Tannic  acid i  gram 

Potassium  alum i 

Distilled  water 40  c.c. 

Dissolve  by  shaking  or  allow  to  stand  overnight  in  the  in- 
cubator. 

II.  Dissolve  "night  blue"  J 0.5  gram 

95  per  cent,  or  absolute  alcohol 20.0  c.c. 

Mix  I  and  II  thoroughly  and  remove  the  heavy  precipitate 
by  filtration.  If  not  used  at  once,  drop  from  a  filter  upon 
the  film.  The  stain  does  not  keep  more  than  a  few  days. 

4.  Wash  carefully  but  thoroughly  in  water. 

5.  Apply  a  saturated  aqueous  solution  of  gentian  violet  for  about 

two  minutes  to  stain  the  bodies  of  the  bacteria. 

6.  Wash  thoroughly  in  water,  dry  with  smooth  blotting-paper, 

and  mount  in  balsam. 

To  secure  a  perfectly  clean  background  for  photomicrography,  it  is 
best  to  stain  on  a  slide.  The  stain  is  then  poured  into  a  Petri  dish,  the 
slide  inverted,  the  end  of  the  slide  used  to  push  aside  the  film  on  the 
surface  of  the  stain,  and  the  film  then  immersed  downward,  one  end  of 
the  slide  supported,  during  staining  on  a  match-stick  or  bit  of  glass  rod. 
In  this  way  the  adherence  of  the  precipitate  to  the  slide  can  be  avoided. 


THE  OBSERVATION  OF  LIVING    PROTOZOA. 

When  protozoa  are  to  be  examined  in  transparent  fluids, 
such  as  pond-water  or  culture  fluids  in  which  they  have  been 
artificially  nourished,  use  can  be  made  of  a  "  live-box  "  or 
of  the  "  hanging  drop."  Ordinarily,  however,  the  organisms 
to  be  examined  are  contained  in  blood,  in  pus,  in  sputum, 
in  feces,  or  in  some  other  more  or  less  opaque  fluid,  of  which 
an  extremely  thin  layer  must  be  prepared  in  order  that  the 

*  "Jour.  Med.  Research,"  vi,  1901,  p.  341. 

t"  Bacteria,"  John  Murray,  London,  2d  edition. 

t  James  Strong  &  Son,  Glasgow  and  Manchester. 


Staining  Protozoa  195 

formed  elements  may  be  separated  sufficiently  for  the  indi- 
vidual cells  and  organisms  to  be  seen. 

Such  a  thin  layer  is  usually  easily  obtained  by  the  use  of 
a  slide  and  cover-glass,  and  the  careful  preparation  of  a  good 
film. 

The  slide  and  the  cover-glass  should  be  thoroughly  cleansed 
and  freed  from  fat  and  grit  and  well  polished.  A  compar- 
atively small  drop  of  blood — let  us  say,  for  example — is 
placed  upon  the  center  of  the  slide  and  immediately  covered 
with  the  cover-glass.  If  the  drop  is  not  too  large  and  the 
glasses  are  clean,  the  weight  of  the  cover-glass  causes  the 
drop  to  spread,  and  capillary  attraction  completes  the  forma- 
tion of  a  very  thin  film.  The  quantity  of  blood  used  should 
not  be  sufficient  to  reach  the  edges  of  the  cover-glass,  else 
sometimes  the  glass  is  pressed  up  instead  of  being  drawn  down 
and  moves  about  too  freely.  If  the  examination  is  to  take 
enough  time  to  cause  the  drop  to  dry,  a  match-stick  dipped  in 
thin  vaselin  and  drawn  about  the  edge  of  the  cover  will 
prevent  it. 

Such  a  film  is  usually  best  examined  at  or  near  the  center, 
where  the  formed  elements  are  most  widely  separatedo 

The  living  protozoa  in  preparations  of  this  kind  may  be 
examined  by  ordinary  illumination  by  transmitted  light,  or 
with  lateral  illumination  by  means  of  the  "  dark-field  il- 
luminator." The  latter  serves  better  for  the  discovery 
of  the  very  small  transparent  organisms — spirochaeta  and 
treponema — and  for  the  observation  of  the  cilia  and  flagella. 

STAINING  PROTOZOA. 

It  is  through  the  study  of  stained  protozoa  that  we  arrive 
at  most  of  our  knowledge  of  their  structural  details.  They 
can  be  stained  in  blood  or  fluids  upon  a  slide  or  in  sections  of 
tissue. 

As  in  the  case  of  the  bacteria,  it  is  first  necessary  to  prepare 
satisfactory  spreads  for  the  purpose.  In  order  that  the 
description  shall  be  as  practical  as  possible,  we  will  suppose 
that  the  micro-organisms  to  be  stained  are  in  blood — spiro- 
chaeta, plasmodium,  etc. 

As  was  pointed  out  above,  the  protozoa,  under  such  circum- 
stances, are  distributed  among  or  in  cellular  elements  that 
interfere  with  satisfactory  observation  unless  precautions  are 
taken  to  separate  them  as  widely  as  may  be  required. 


196       Methods  of  Observing  Micro-organisms 

i.  Cover-glasses. — The  glasses  should  be  perfectly  clean  and  freed 
from  fat,  either  by  washing  in  alcohol  and  ether  and  wiping  with 
a  clean  soft  cotton  cloth  or  Chinese  rice  paper,  or  by  flaming. 
The  drop  of  blood  should  be  small  and  should  be  placed 
upon  the  center  of  one  glass  and  immediately  covered  by 
another,  so  held  that  the  corners  do  not  coincide.  As  soon 
as  the  drop  is  fairly  well  distributed  the  glasses  are  gently 
slid  apart. 


Fig. 


36. — Method  of   making  dry  film  with  two  cover-glasses  (from 
Daniels'  "  Laboratory  Studies  in  Tropical  Medicine"). 


Slides. — The  slides,  like  the  cover-glasses,  must  be  perfectly 
clean.  The  drop  of  blood  is  placed  upon  one  slide  at  about 
one-fourth  the  length  of  the  slide  from  its  end,  touched  with 
the  end  (it  must  have  ground  edges)  of  the  second  slide,  and 
then  gently  pushed  along  until  the  fluid  is  exhausted. 


Fig.  37- — Method  of  making  dry  films  with  two  slides  (from  Daniels' 
"  Laboratory  Studies  in  Tropical  Medicine  "). 


Staining  Protozoa  197 

If  the  covers  are  to  be  stained,  they  can  most  conveniently 
be  held  in  the  Stewart  forceps.  If  the  slides  are  used,  they 
can  be  held  in  the  fingers. 

The  stain  most  useful  is  that  of  Romanowsky.  It  has 
many  modifications,  of  which  the  most  used  and  best  known 
are  Giemsa's,  Jenner's,  Leishmann's,  Wright's,  and  Marino's. 
These  stains  can  be  bought  either  in  solution  or  in  tablet 
form  ready  for  solution. 

Those  most  highly  to  be  recommended  are  Wright's  and 
Marino's. 

Wright's  Blood-stain. — This  is  a  modification  of  Leishmann's  stain, 
to  which  it  is  to  be  preferred  because  it  can  be  made  in  a  few 
hours  instead  of  eleven  days.  It  combines  the  methylene-blue- 
eosin  combination  of  Romanowsky  with  the  methyl -alcohol 
fixation  of  Jenner. 

It  is  prepared  as  follows:* 

''To  a  0.5  per  cent,  aqueous  solution  of  sodium  bicarbonate  add 
methylene-blue  (B.  X.  or  "medicinally  pure")  in  the  proportion 
of  i  gm.  of  the  dye  to  100  c.c.  of  the  solution.  Heat  the  mixture  in 
a  steam  sterilizer  at  100°  C.  for  one  full  hour,  counting  the  time 
after  the  sterilizer  has  become  thoroughly  heated.  The  mixture 
is  to  be  contained  in  a  flask  of  such  size  and  shape  that  it  forms 
a  layer  not  more  than  6  cm.  deep.  After  heating,  the  mixture  is 
allowed  to  cool,  placing  the  flask  in  cold  water  if  desired,  and  is 
then  filtered,  to  remove  the  precipitate  which  has  formed  in  it. 
It  should,  when  cold,  have  a  deep  purple-red  color  when  viewed, 
in  a  thin  layer,  by  transmitted  yellowish  artificial  light.  It 
does  not  show  this  color  while  it  is  warm.  To  each  100  c.c. 
of  the  filtered  mixture  add  500  c.c.  of  a  o.i  per  cent,  aqueous 
solution  of  "yellowish,  water-soluble"  eosin  and  mix  thoroughly. 
Collect  the  abundant  precipitate  which  immediately  appears 
on  a  filter.  When  the  precipitate  is  dry,  dissolve  it  in  methylic 
alcohol  (Merck's  "reagent")  in  the  proportion  of  o.i  gr.  to 
60  c.c.  of  the  alcohol.  In  order  to  facilitate  the  solution  the 
precipitate  is  to  be  rubbed  up  with  the  alcohol  in  a  porcelain 
dish  or  mortar  with  a  spatula  or  pestle. 

"This  alcoholic  solution  of  the  precipitate  is  the  staining  fluid.  It 
should  be  kept  in  a  well-stoppered  bottle  because  of  the  volatility 
of  the  alcohol.  If  it  becomes  too  concentrated  by  evaporation, 
and  thus  stains  too  deeply  or  forms  a  precipitate  on  the  blood- 
smear,  the  addition  of  a  suitable  quantity  of  methylic  alcohol  will 
quickly  correct  such  fault.  It  does  not  undergo  any  other  spon- 
taneous change  than  that  of  concentration  by  evaporation." 

Method  of  Staining. — The  blood-films  are  permitted  to  dry  in  the  air 
(not  heated) : 

1.  Cover  the  film  with  a  noted  quantity  of  the  staining  fluid  by 

means  of  a  medicine  dropper. 

2.  After  one  minute  add  to  the  staining  fluid  the  same  quantity  of 

distilled  water  by  means  of  the  medicine  dropper,  and  allow  it 

*Mallory  and  Wright,  "Pathological  Technique,"  1911,  p.  364. 


198       Methods  of  Observing  Micro-organisms 

to  remain  for  two  or  three  minutes,  according  to  the  intensity 
of  the  staining  desired.  A  longer  period  of  staining  may  pro- 
duce a  precipitate. 

3.  Wash  the  preparation  in  water  for  thirty  seconds  or  until  the 

thinner  portions  of  the  preparation  become  yellow  or  pink  in 
color. 

4.  Dry  and  mount  in  balsam. 

Films  more  than  an  hour  old  do  not  stain  so  well  as  fresh  ones.  Old 
films  show  bluish  instead  of  pink  erythrocytes. 

Marino's  stain*  is  extremely  delicate  and  gives  still  more  beautiful 
results  where  parasites  are  present.  It  is  an  azur-eosin  combination, 
prepared  as  follows: 

Solution  I: 

Methylene-blue  (medicinal) 0.5  gram. 

Azur  II 0.5      " 

Water  (distilled) 100.0  grams. 

Solution  II: 

Sodium  carbonate 0.5  gram. 

Water 100.0  c.c. 

Pour  the  two  solutions  together  and  stand  the  mixture  in  the 
thermostat  for  forty-eight  hours  at  37°  C.;  then  add  0.2  per 
cent,  aqueous  solution  of  eosin  ("yellowish  aqueous  eosin"). 
The  quantity  of  this  solution  must  be  varied  according  to  the 
blue  dyes  employed,  so  as  to  secure  the  maximum  precipitation. 
The  exact  quantity  can  only  be  determined  by  titration.  A  pre- 
cipitate now  forms  in  the  course  of  twenty-four  hours.  This  is 
caught  upon  a  filter-paper  and  dried. 

The  precipitate,  dissolved  in  methylic  alcohol,  in  the  proportion  of 
0.04  gm.  of  the  powder  to  20  c.c.  of  the  methylic  alcohol,  forms 
the  stain. 

Method. — The  stain  is  dropped  upon  the  spread  so  as  to  cover  it, 
the  number  of  drops  being  counted.  It  is  permitted  to  act  for 
exactly  three  minutes  for  purposes  of  fixation,  then,  without  pour- 
ing off  the  stain,  twice  the  number  of  drops  of  a  i :  100,000  aqueous 
eosin  solution  are  added.  The  two  fluids  gradually  mix,  trans- 
fusion currents  are  formed,  and  the  specimen  is  allowed  to  stand 
for  exactly  two  minutes  longer.  It  is  during  this  time  that  the 
staining  takes  place.  A  precipitate  usually  forms  upon  the 
surface  of  the  fluid,  so  that  it  must  not  be  poured  off,  but  splashed 
off  by  dropping  distilled  water  upon  it  from  a  height.  The  dis- 
tilled water  is  added  until  it  no  longer  shows  any  color,  when  the 
specimen  is  drained,  dried,  and  mounted  in  balsam. 

The  student  may  also  try  staining  with  hematoxylon  and 
eosin,  thionin  and  eosin,  methylene-blue  and  eosin,  or  any 
other  dyes,  some  of  which  sometimes  bring  out  special  de- 
tails of  structure.  The  protozoa  do  not  show  the  same  re- 
action to  Gram's  stain  that  makes  it  so  useful  for  differ- 
entiating the  bacteria. 

*  "Ann.  de  1'Inst.  Pasteur,"  1904,  xvm,  761. 


Staining  Protozoa  in  Tissue  199 

STAINING  PROTOZOA  IN  TISSUE. 

For  this  purpose  the  sections  should  be  embedded  in  paraf- 
fin, cut  very  thin,  and  cemented  to  the  slides. 

Ordinary  staining  with  hematoxylin  and  eosin  is  rarely  of 
much  use.  Methylene-blue  and  eosin  is  better,  but  still 
more  useful  are  the  Romanowsky  methods,  and  both  the 
Wright  stain  and  the  Marino  stain  can,  with  some  modifica- 
tion of  the  time  of  staining  and  washing,  be  employed  with 
good  results. 

Still  better  and  more  satisfactory  for  certain  protozoa  are 
the  iron-hematoxylin  and  the  Biondi  stain. 

Heidenhain's  Iron-hematoxylin.* — Fix  the  tissue,  by  preference,  in 
Zenker's  solution,  though  alcohol  fixation  will  do.  Embed  in 
paraffin,  cut  very  thin,  and  fix  to  the  slide. 

1.  Stain  from  three  to  twelve  hours  in  2.5  per  cent,  solution  of 
violet  iron-alum  (sulphate  of   iron  and  ammonium).     The  sec- 
tions should  be  stood  vertically  in  the  solution,  so  that  no  pre- 
cipitate may  form  upon  them. 

2.  Wash  quickly  in  water. 

3.  Stain  in  a  0.5  per  cent,  ripened  alcoholic  solution  of  hematoxylin 
for  from  twelve  to  thirty-six  hours. 

4.  Wash  in  water. 

5.  Differentiate  in  the  iron-alum  solution,  controlling  the  results 
under  the  microscope.     The  section  should  be  well  washed  in  a 
large  dish  of  tap-water  before  each  examination  to  stop  decoloriza- 
tion. 

6.  Wash  in  running  water  for  a  quarter  of  an  hour. 

7.  Pass  through  alcohol,  xylol,  and  mount  in  xylol  balsam. 

A  counterstain  with  Bordeau  R.  before  or  with  rubin  S.  after  the 
iron  stain  is  sometimes  useful. 

Biondi-Heidenhain  Stain* — The  tissues  must  be  fixed  in  Zenker's 
or  corrosive  sublimate  solutions.  Embed  in  paraffin,  cut  very 
thin,  fix  to  the  slide. 

Stain      I.  Orange  G 8  grams. 

Water 100 

II.  Acid  fuchsin  \ 

or  Rubin  S.   J 

Water 100 

III.  Methyl-green 8  grams. 

Water 100 

Let  the  solutions  stand  for  several  days,  occasionally  shaking 
the  bottles  to  make  sure  that  a  saturated  solution  of  each  is 
secured.  At  the  end  of  the  time  set,  mix  the  solutions  in  the 
following  preparations : 

1 100  parts. 

II 20 

III 50 

*  Mallory  and  Wright,  "Pathological  Technique,"  1911,  p.  309- 
f  Modified  from  Mallory  and  Wright,  "  Pathological  Technique," 
1911,  p.  289. 


200       Methods  of  Observing  Micro-organisms 

At  the  time  of  staining  dilute  the  mixture  i :  60  or  i :  100  with 
water.  To  test  the  solution :  ( i )  Acetic  acid  makes  it  redder. 
(2)  A  drop  of  the  solution  on  filter-paper  should  make  a  blue 
spot  with  a  green  center  and  an  orange  border.  If  a  red 
zone  appears  outside  of  the  orange,  too  much  acid  fuchsin 
is  present. 

1 .  Stain  the  sections  from  six  to  twenty-four  hours. 

2.  Wash  out  a  little  in  90  per  cent,  alcohol. 

3.  Dehydrate  in  absolute  alcohol. 

4.  Xylol. 

5.  Xylol  balsam. 

It  is  important  to  place  the  sections  directly  from  the  staining  fluid 
into  the  alcohol,  because  water  washes  out  the  methyl-green 
instantly. 

Ross'  Thick  Blood-spreads. — In  case  the  number  of  parasites  in  the 
blood  is  very  small,  so  that  they  would  be  scattered  sparingly 
over  a  large  area  of  the  ordinary  blood  spread,  Ross*  has  suggested 
a  modification  of  the  technic  by  which  they  can  be  more  readily 
found.  To  do  this  a  very  thick  spread  is  prepared  and  dried. 
As  soon  as  it  is  dry,  and  without  fixing,  the  slide  is  stood  vertically 
in  a  vessel  filled  with  distilled  water.  The  red  corpuscles  at 
once  begin  to  hemolyze  and  the  process  is  carried  on  to  com- 
pletion. When  all  of  the  hemoglobin  has  been  removed,  the 
slide  is  taken  out,  dried,  and  then  fixed  and  stained.  There 
now  being  no  red  corpuscles  to  distract  the  attention  or  obscure 
the  vision,  the  stained  parasites  can  quickly  be  found. 

Measurement  of  Micro-organisms. — They  can  best  be 
measured  by  an  eyepiece  micrometer.  As  these  instruments 
vary  somewhat  in  construction,  the  unit  of  measurement 
for  each  objective  magnification  and  the  method  of  manipu- 
lating the  instruments  must  be  learned  from  dealers'  cata- 
logues. 

Photographing  Microorganisms.— This  requires  special 
apparatus  and  methods,  for  which  it  is  necessary  to  refer 
to  special  text-books. f 

*  "Lancet,"  Jan.  10,  1903. 

f  See  the  excellent  chapter  upon  Photomicrography  in  Aschoff  and 
Gaylord's  "Pathological  Histology,"  Philadelphia,  1900. 


CHAPTER   VI. 

STERILIZATION  AND  DISINFECTION* 

BEFORE  beginning  the  consideration  of  the  methods  em- 
ployed for  the  artificial  cultivation  of  individual  micro- 
organisms and  the  preparation  of  media  for  that  purpose,  it 
is  necessary  to  have  a  thorough  knowledge  of  the  principles 
of  sterilization  and  disinfection  in  order  intelligently  to  ap- 
ply the  methods  employed  for  the  elimination  or  destruction 
of  others  whose  accidental  presence  might  ruin  our  experi- 
ments. 

The  dust  of  the  atmosphere,  almost  invariable  in  its  micro- 
organismal  contamination,  constantly  settles  upon  our  glass- 
ware, pots,  kettles,  funnels,  etc.,  and  would  certainly  ruin 
every  culture-medium  with  which  we  experiment  did  we  not 
take  appropriate  measures  for  its  purification  and  protection. 

To  get  rid  of  these  undesirable  "  weeds  "  we  make  use  of 
our  knowledge  of  the  conditions  destructive  to  bacterial  life, 
and  subject  the  articles  contaminated  by  them  to  the  action 
of  heat  beyond  their  known  enduring  power,  or  to  the  action 
of  chemic  agents  known  to  destroy  them,  or  remove  them 
from  fluids  into  which  they  have  entered  by  passage  through 
unglazed  porcelain.  By  all  of  these  methods  the  articles 
are  made  sterile.  Anything  is  sterile  when  it  contains  no 
germs  of  life. 

Sterilization  is  the  act  of  making  sterile  by  destroying  or 
removing  all  micro-organismal  life,  whether  infectious  or 
non-infectious.  Disinfection  signifies  the  destruction  of  the 
infectious  agents,  taking  no  account  of  those  that  are  non- 
infectious.  A  germicide  is  any  substance  that  will  kill 
germs.  It  may  be  used  for  disinfection  and  for  sterilization. 
An  antiseptic  is  a  substance  that  will  inhibit  the  growth  of 
micro-organisms.  It  does  not  necessarily  kill  them. 

The  table  on  page  202  will  serve  to  outline  the  methods 
used  for  effecting  sterilization  or  the  complete  destruction 
or  removal  of  living  organisms: 

201 


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Sterilization  and  Disinfection 


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Methods  of  Sterilization 


203 


I.  The  Sterilization  and  Protection  of  Instruments 
and  Glassware. — Sterilization  may  be  accomplished  by 
either  moist  or  dry  heat.  For  the  perfect  sterilization  of 
objects  capable  of  withstanding  it  dry  heat  is  always  to  be 
preferred,  because  of  its  more  certain  action.  If  we  knew 
just  what  organisms  we  had  to  deal  with,  we  might  be  able 
in  many  cases  to  save  time  and  gas ;  but  though  some  non- 
spore-producing  forms  are  killed  at  a  temperature  of  60°  C., 


pig  3g — Hot-air  sterilizer.  The  gas  jets  are  inclosed  within  the 
space  between  the  outer  and  middle  walls,  C,  and  can  be  seen  at  F. 
The  heat  ascends,  warming  the  air  between  the  two  Inner  walls,  which 
ascends  between  the  walls,  K,  then  descends  over  the  contents,  /,  and 
escapes  through  perforations  in  the  bottom,  B,  to  supply  the  draft  at 
F,  and  eventually  escapes  again  at  S;  R,  gas  regulator;  T,  thermometer. 

spore-bearers  may  withstand  100°  C  for  an  hour;  it  is, 
therefore,  best  to  employ  a  temperature  high  enough  to  kill 
all  with  certainty.  The  sterilizing  apparatus,  or  "hot-air 
sterilizer,"  is  shown  in  Fig.  38. 

Platinum  wires  used  for  inoculation  are  sterilized  by  being 
held  in  the  direct  flame  until  they  become  incandescent. 


204  Sterilization  and  Disinfection 

In  sterilizing  the  wires  attention  must  be  bestowed  upon 
the  glass  handle,  which  should  be  flamed  for  at  least  half 
its  length  for  a  few  moments  when  used  for  the  first  time 
each  day.  Carelessness  in  this  respect  may  result  in  the 
contamination  of  the  cultures. 

Knives,  scissors,  and  forceps  may  be  exposed  for  a  very 
brief  time  to  the  direct  flame,  but  as  this  affects  the  temper 
of  the  steel  when  continued  too  long,  they  are  better  boiled, 
steamed,  or  carbolized. 

All  articles  of  glassware  are  to  be  sterilized  by  an  exposure 
of  one-half  to  one  hour  to  a  sufficiently  high  temperature — 
150°  C.  or  302°  F. — in  an  appropriate  hot-air  oven.  This 
temperature  is  fatal  to  all  forms  of  microscopic  life. 

Rubber  stoppers,  corks,  wooden  apparatus,  and  other 
objects  which  are  warped,  cracked,  charred,  or  melted 
by  so  high  a  temperature  must  be  sterilized  by  exposure  to 
streaming  steam  or  steam  under  pressure,  in  the  steam 
sterilizer  or  autoclave,  before  they  can  be  pronounced 
sterile. 

It  must  always  be  borne  in  mind  that  after  sterilization 
has  been  accomplished  the  original  sources  of  contamination 
are  still  present,  so  that  it  is  necessary  to  protect  the  sterilized 
objects  and  media  from  them. 

To  Schroder  and  Van  Dusch  belongs  the  credit  of  having 
first  shown  that  when  the  mouths  of  flasks  and  tubes  are 
closed  with  plugs  of  sterile  cotton  no  germs  can  filter  through. 
This  discovery  has  been  of  inestimable  value,  and  has  been 
one  of  the  chief  means  permitting  the  advance  of  bacteri- 
ology. If,  before  sterilizing,  flasks  and  tubes  are  carefully 
plugged  with  ordinary  (non-absorbent)  cotton-wool,  they  and 
their  contents  will  remain  free  from  the  access  of  germs  until 
opened.  Instruments  may  be  sterilized  wrapped  in  cotton, 
to  be  opened  only  when  ready  for  use ;  or  instruments  and 
rubber  goods  sterilized  by  steam  can  subsequently  be 
wrapped  in  sterile  cotton  and  kept  for  use.  It  is  of  the 
utmost  importance  to  carefully  protect  every  sterilized 
object,  in  order  that  the  object  of  the  sterilization  be  not 
defeated.  As  the  spores  of  molds  falling  upon  cotton  some- 
times grow  and  allow  their  mycelia  to  work  their  way 
through  and  drop  into  the  culture-medium.  Roux  has  em- 
ployed paper  caps,  with  which  the  cotton  stoppers  can  be 
protected  from  the  dust.  These  are  easily  made  by  curling  a 
small  square  of  paper  into  a  "  cornucopia,"  and  fastening  by 


Methods  of  Sterilization 


205 


turning  up  the  edge  or  putting  in  a  pin.  The  paper  is  placed 
over  the  stopper  before  the  sterilization,  after  which  no 
contamination  of  the  cotton  can  occur. 

II.  Sterilization  and  Protection  of  Culture=media. — 
As  almost  all  of  the  culture-media  contain  about  80  per 
cent,  of  water,  which  would  evaporate  in  the  hot-air  closet, 
and  so  destroy  the  material,  hot-air  sterilization  is  inap- 
propriate for  them,  sterilization  by  streaming  steam  being 
the  only  satisfactory  method.  The  prepared  media  are 


Fig-  39- — Arnold's  steam  sterilizer  (Boston  Board  of  Health  form). 


placed  in  previously  sterilized  flasks  or  tubes,  carefully 
plugged  with  cotton- wool,  and  then  sterilized  in  an  Arnold's 
steam  sterilizer. 

The  temperature  of  boiling  water,  100°  C.,  does  not  kill 
the  spores,  so  that  one  exposure  of  the  culture-media  to 
streaming  steam  is  of  little  use.  The  sterilization  must  be 
applied  in  a  systematic  manner — intermittent  sterilization — 
based  upon  a  knowledge  of  sporulation. 

In  carrying  out  intermittent  sterilization  the  culture- 
medium  is  exposed  for  fifteen  minutes  to  the  passage  of 


206 


Sterilization  and  Disinfection 


streaming  steam  or  to  some  temperature  judged  to  be 
sufficiently  high,  so  that  the  adult  micro-organisms  con- 
tained in  it  are  killed.  As  the  spores  remain  uninjured, 
the  medium  is  stood  aside  in  a  cool  place  for  twenty -four 
hours,  and  the  spores  allowed  slowly  to  develop  into  adult 
organisms. 

When  the  twenty-four  hours  have  passed,  the  medium  is 
again  exposed  to  the  same  temperature  until  these  newly 
developed  bacteria  are  also  killed.  Eventually,  the  process 

is  repeated  a  third  time,  lest  a  few 
spores  remain  alive.  When  prop- 
erly sterilized  in  this  way  culture- 
media  will  remain  free  from  con- 
tamination indefinitely. 

In  popular  parlance,  the  inter- 
mittent exposure  of  the  culture- 
media  to  steam  is  spoken  of  as 
sterilization. 

A  prolonged  single  exposure  to 
lower  temperatures  (6o0-yo0  C.), 
known  as  pasteurization,  is  em- 
ployed for  the  destruction  of  bac- 
teria in  milk  and  other  fluids  that 
are  injured  or  coagulated  by  ex- 
posure to  100°  C.  It  is  appro- 
priate only  when  the  organisms  to 
be  killed  are  without  spores  and 
without  marked  resisting  powers. 

Sterilization  in  the  Autoclave. — 
If  it  should  be  desirable  to  sterilize 
a  medium  at  once,  not  waiting  the 
three  days   required  by  the  inter- 
mittent method,  it   may  be   done 
by    superheated    steam    under    a 
pressure    of    two    or    three    atmo- 
spheres, sufficient  heat  being  generated  to  immediately  de- 
stroy the  spores. 

Because  of  its  convenience  many  laboratory  workers 
habitually  use  the  autoclave  for  the  sterilization  of  all 
media  not  injured  by  the  high  temperature.  The  steriliza- 
tion, to  be  complete,  requires  that  the  exposure  shall  be 
for  fifteen  minutes  at  110°  C.  (six  pounds'  pressure). 


Fig.  40. — Modern  auto- 
clave. 


Sterilization  in  the  Autoclave 


207 


The  media  to  be  sterilized  should  be  placed  in  the  autoclave,  the  top 
firmly  screwed  down,  but  the  escape-valve  allowed  to  remain  open 
until  steam  is  freely  generated  within  and  replaces  the  hot  air.  The 
valve  is  then  closed,  and  the  temperature  maintained  for  fifteen  min- 
utes or  longer  if  the  media  be  in  bulk  in  flasks.  The  apparatus  should 


Fig.  41. — Pasteur-Chamberland  filter  arranged  to  filter  under 
pressure. 

be  permitted  to  cool  before  the  valve  is  opened,  and  the  vacuum  be 
slowly  relieved  If  the  valve  be  opened  suddenly  the  fluids  boil  rap- 
idly and  the  cotton  plugs  may  be  forced  into  the  tubes  or  flasks  by 
the  air  pressure.  The  chief  objection  to  the  use  of  the  autoclave  is  that 
the  high  temperature  sometimes  brings  about  chemic  changes  in  the 
media  by  which  the  reaction  is  altered. 


208 


Sterilization  and  Disinfection 


Sterilization  by  Filtration. — Liquids  that  cannot  be 
subjected  to  heat  without  the  loss  of  their  most  important 
qualities  may  be  sterilized  by  nitration — i.  e.,  by  passing 
them  through  unglazed  porcelain  or  some  other  material 
whose  interstices  are  sufficiently  fine  to  resist  the  passage 
of  bacteria.  This  method  is  largely  employed  for  the 
sterilization  of  the  unstable  bacterial  toxins  that  are  de- 
stroyed by  heat.  Various  substances  have  been  used  for 
nitration,  as  diatomaceous  earth  (Berkefeld  filters),  stone, 


Fig.   42. — Different    types    of    bacteriologic    filters  :    a,    Kitasato  ;    b, 
Berkefeld;  c,  Chamberland ;  d,  Reichel. 

sand,  powdered  glass,  etc.,  but  experimentation  has  shown 
unglazed  porcelain  to  be  the  only  reliable  filtering  material 
by  which  to  remove  bacteria.  Even  this  material,  whose 
interstices  are  so  small  as  to  allow  the  liquid  to  pass  through 
with  great  slowness,  is  only  certain  in  its  action  for  a  time, 
for  after  it  has  been  repeatedly  used  the  bacteria  seem 
able  to  work  their  way  through.  To  be  certain  of  the  efficacy 
of  any  filter,  the  fluid  first  passed  through  must  be  tested 
by  cultivation  methods  to  prove  that  all  the  bacteria  have 
been  removed.  The  complicated  Pasteur-Chamberland  and 
the  simple  Kitasato  and  Reichel  filters  are  shown  in  figures 
41  and  42. 


Disinfection  of  the  Hands  209 

The  porcelain  bougies  as  well  as  their  attachments  must 
be  thoroughly  sterilized  before  use. 

After  having  been  used,  a  porcelain  filter  must  be  dis- 
infected, scrubbed,  dried  thoroughly,  and  then  heated  in 
a  Bunsen  burner  or  blowpipe  flame  until  all  the  organic 
matter  is  consumed.  In  this  firing  process  the  filter  first 
turns  black  as  the  organic  matter  chars,  then  becomes 
white  again  as  it  is  consumed.  The  porcelain  must  be  dry 
before  entering  the  fire,  or  it  is  apt  to  crack. 

It  should  not  be  forgotten  that  the  filtrate  must  be  received 
in  sterile  receivers  and  handled  with  care  to  prevent  sub 
sequent  contamination. 

The  filtration  of  water,  peptone  solution,  and  bouillon 
is  comparatively  easy,  but  gelatin  and  blood-serum  pass 
through  with  great  difficulty,  and  speedily  gum  the  filter. 

III.  The  Disinfection  of  Instruments,  Ligatures, 
Sutures,  the  Hands,  etc. — There  are  certain  objects  used 
by  the  surgeon  that  cannot  well  be  rendered  incandescent, 
exposed  to  dry  heat  at  150°  C.,  or  steamed  continuously,  or 
intermittently  heated  without  injury.  For  these  objects  dis- 
infection must  be  practised.  Ever  since  Sir  Joseph  Lister 
introduced  antisepsis,  or  disinfection,  into  surgery  there  has 
been  a  struggle  for  the  supremacy  of  this  or  that  highly  rec- 
ommended germicidal  substance,  with  two  results — viz.,  that 
a  great  number  of  feeble  germicides  have  been  discovered, 
and  that  belief  in  the  efficacy  of  all  germicides  has  been 
somewhat  shaken;  hence  the  aseptic  surgery  of  the  present 
day,  which  strives  to  prevent  the  entrance  of  germs  into  the 
wound  rather  than  their  destruction  after  admission  to  it. 

For  a  complete  discussion  of  the  subject  of  antiseptics 
in  relation  to  surgery  the  reader  must  be  referred  to  text- 
books of  surgery. 

The  Disinfection  of  the  Hands,  etc. — The  disinfection  of 
the  skin — both  the  hands  of  the  surgeon  and  the  part  about 
to  be  incised — is  a  matter  of  the  utmost  importance.  Wash- 
ing the  hands  with  soap,  which  has  marked  germicidal 
properties,  will  in  many  cases  suffice  to  destroy  or  remove 
bacteria  from  smooth  skins.  This  method,  which  is  regarded 
by  some  surgeons  as  adequate,  is  not,  however,  commonly 
regarded  as  sufficient  protection  to  the  patient  who  might 
be  infected  by  any  remaining  micro-organisms.  To  over- 
come this,  many  surgeons  prefer  the  use  of  sterilized  gloves 

14 


210  Sterilization  and  Disinfection 

of  thin  rubber  to  all  other  means  of  preventing  manual  in- 
fections. Others  prefer  to  use  detergent  and  disinfectant 
measures.  The  method  at  present  generally  employed,  and 
recommended  by  Welch  and  Hunter  Robb,  is  as  follows: 
The  nails  must  be  trimmed  short  and  perfectly  cleansed. 
The  hands  are  washed  thoroughly  for  ten  minutes  in  water 
of  as  high  a  temperature  as  can  comfortably  be  borne,  soap 
and  a  previously  sterilized  brush  being  freely  used,  and 
afterward  the  excess  of  soap  washed  off  in  clean  hot  water. 
The  hands  are  then  immersed  for  from  one  to  two  minutes 
in  a  warm  saturated  solution  of  permanganate  of  potassium, 
then  in  a  warm  saturated  solution  of  oxalic  acid,  until 
complete  decolorization  of  the  permanganate  occurs,  after 
which  they  are  washed  free  from  the  acid  in  clean  warm 
water  or  salt  solution.  Finally,  they  are  soaked  for  two 
minutes  in  a  i :  500  solution  of  bichlorid  of  mercury. 

Lockwood,*  of  St.  Bartholomew's  Hospital,  recommends, 
after  the  use  of  the  scissors  and  penknife,  scrubbing  the 
hands  and  arms  for  three  minutes  in  hot  water  and  soap 
to  remove  all  grease  and  dirt.  The  scrubbing  brush  ought 
to  be  steamed  or  boiled  before  use,  and  kept  in  i  :  1000 
biniodid  of  mercury  solution.  When  the  soapsuds  have 
been  thoroughly  washed  away  with  plenty  of  clean  water, 
the  hands  and  arms  are  thoroughly  washed  and  soaked 
for  not  less  than  two  minutes  in  a  solution  of  biniodid  of 
mercury  in  methylated  spirit;  i  part  of  the  biniodid  in  500 
of  the  spirit.  Hands  that  cannot  bear  i  :  1000  bichlorid 
and  5  per  cent,  carbolic  solutions  bear  frequent  treatment 
with  the  biniodid.  After  the  spirit  and  biniodid  have  been 
used  for  not  less  than  two  minutes,  the  solution  is  washed 
off  in  i  :  2000  or  i  :  4000  biniodid  of  mercury  solution. 

It  is  a  mistake  to  insist  upon  the  employment  of  disinfect- 
ing solutions  of  a  strength  injurious  to  the  skin.  It  must  be 
obvious  to  every  one  that  rough  skins  with  numerous  hang- 
nails and  fissures  offer  greater  difficulties  to  be  overcome  in 
disinfection,  and  more  readily  convey  micro-organisms  into 
the  wound  than  smooth,  soft  skins. 

Sterilization  of  Ligatures,  etc.— Catgut  cannot  be  steril- 
ized by  boiling  without  deterioration.  The  present  method 
of  treatment  is  to  dry  it  in  a  hot-air  chamber  and  then  boil  it 
in  cumol,  which  is  afterward  evaporated  and  the  skeins  pre- 
*  "  Brit.  Med.  Jour.,"  July  u,  1896. 


Disinfection  of  Sick-chambers,  etc.  211 

served  in  sterile  test-tubes  or  special  receptacles  plugged  with 
sterile  cotton.  Cumol  was  first  introduced  for  this  purpose 
by  Kronig,  as  its  boiling-point  is  i68°-i78°  C.,  and  thus 
sufficiently  high  to  kill  spores.  The  use  of  cumol  for  the 
sterilization  of  catgut  has  been  carefully  investigated  by 
Clarke  and  Miller.* 

Catgut  may  also  and  equally  well  be  sterilized  by  the  use 
of  chemical  agents.  This  subject  has  been  carefully  reviewed 
by  Bertarelli  and  Bocchia,f  who  regard  the  method  of 
Claudius  and  the  modification  of  it  by  Rogone  as  the  best. 
The  method  of  Claudius  is  to  roll  the  catgut  into  skeins  and, 
without  taking  any  precautions  to  remove  any  fat  it  may  con- 
tain, place  it  in  a  mixture  of  iodin  i,  iodid  of  potassium  i,  and 
distilled  water  100.  After  immersion  for  eight  days  the  cat- 
gut is  removed,  under  aseptic  precautions,  to  alcohol  or  to 
3  per  cent,  carbolic  solution,  in  which  it  is  indefinitely  pre- 
served for  use. 

Ligatures  of  silk  and  silkworm  gut  are  boiled  in  water 
immediately  before  using,  or  are  steamed  with  the  dressings, 
or  placed  in  test-tubes  plugged  with  cotton  and  steamed  in 
the  sterilizer. 

Sterilization  of  Surgical  Instruments,  etc. — In  most 
hospitals  instruments  are  boiled,  before  using,  in  a  i  to  2  per 
cent,  soda  (sodium  carbonate,  sodium  bicarbonate,  or  sodium 
biborate)  solution,  as  plain  water  has  the  disadvantage  of 
rusting  them.  During  the  operation  they  are  either  kept  in 
the  boiled  water  or  in  a  carbolic  solution,  or  are  dried  with  a 
sterile  towel.  Andrews  makes  special  mention  of  the  fact 
that  the  instruments  must  be  completely  immersed  to  pre- 
vent rusting. 

Disinfection  of  the  Wound. — Cleansing  solutions  (normal 
salt  solution)  and  disinfecting  solutions  (such  as  i :  10,000  to 
i :  1000  bichlorid  of  mercury)  are  only  applied  to  septic 
wounds. 

IV.  The  Disinfection  of  Sick=chambers,  Dejecta,  etc. 
—The  Air  of  the  Sick-room. — It  is  impossible  to  sterilize 
or  disinfect  the  atmosphere  of  a  room  during  its  occupancy 
by  the  patient.  It  is  entirely  useless  to  place  beneath  the  bed 
or  in  the  corner  of  a  room  small  receptacles  filled  with  car- 
bolic acid  or  chlorinated  lime.  These  can  serve  no  purpose 

*  "Bull,  of  the  Johns  Hopkins  Hospital,"  Feb.  and  March,  1896. 

t  "Centralbl.  fur  Bakt.  u.  Parasitenk.,"  Orig.  i,,  620. 


212  Sterilization  and  Disinfection 

for  good,  and  may  do  harm  by  obscuring  odors  emanating 
from  harmful  materials  that  should  be  removed  from  the 
room.  The  practice  is  only  comparable  to  the  old  faith  in 
the  virtue  of  asafetida  tied  in  a  corner  of  the  handkerchief 
as  a  preventive  of  cholera  and  smallpox. 

Before  one  is  able  to  make  a  scientific  application  of  any 
germicidal  substance  it  is  necessary  to  become  acquainted  with 
its  micro-organism-destroying  powers.  This  may  seem  at 
first  thought  to  be  a  simple  matter,  but  is,  in  reality,  one  of 
great  complexity  and  difficulty,  for  the  various  micro-organ- 
isms show  marked  variations  in  their  powers  of  endurance; 
different  stages  in  the  development  of  the  micro-organisms 
show  different  degrees  of  resisting  power,  and  the  conditions 
under  which  the  germicide  meets  the  micro-organism  effect 
marked  variations  in  action.  These  factors  make  it  neces- 
sary to  vary  the  process  of  disinfection  according  to  the  exact 
purpose  to  be  achieved. 

Let  two  examples  serve  to  illustrate  these  requirements: 
Bichlorid  of  mercury  is  one  of  the  most  powerful,  reliable,  and 
generally  useful  germicides,  but  the  strength  of  its  solutions 
must  vary  according  to  the  purpose  for  which  they  are 
intended.  It  kills  cocci  and  non-sporogenic  bacilli  in  dilu- 
tions of  i :  10,000  in  from  five  minutes  to  twenty-four  hours, 
but  to  kill  anthrax  spores  requires  twenty-four  hours'  immer- 
sion in  i :  2000  solution.  If  albuminous  substances  are 
present  in  the  medium  containing  the  micro-organisms  they 
precipitate  the  salt  immediately,  diminishing  the  strength  of 
the  solution  and  so  retarding  or  perhaps  preventing  the 
germicidal  action.  Again,  certain  micro-organisms  are  de- 
fended from  the  action  of  destructive  agents,  and  among  them 
the  germicides,  through  the  presence  of  waxy  matter  in  their 
substance.  Such  is  the  case  with  the  acid-fast  organisms, 
and  notably  the  tubercle  bacillus.  Antiformin,  a  combina- 
tion composed  of  equal  parts  of  liquor  sodae  chlorinatae  and 
a  15  per  cent,  solution  of  caustic  soda,  immediately  dis- 
solves the  great  majority  of  micro-organisms,  but  has  no 
destructive  action  whatever  upon  the  tubercle  bacillus. 

The  most  useful  germicidal  substances  act  destructively 
upon  the  micro-organisms  by  forming  chemical  compounds 
with  their  cytoplasm.  Thus,  the  salts  of  mercury  unite  with 
the  protoplasm  to  form  an  albuminate  of  mercury.  Other 
germicidal  agents  dissolve  or  coagulate  the  protoplasm ;  still 
others  oxidize  and  so  completely  destroy  the  cells.  In  the 


Inorganic  Disinfectants  213 

process  of  germicidal  action  many  and  varied  activities  are 
at  work,  and,  as  all  are  not  understood,  the  subject  is  a 
difficult  one  to  handle  in  a  limited  amount  of  space.  With 
the  salts,  acids,  and  bases  it  appears  from  the  researches  of 
Kronig  and  Paul*  that  ionization  in  solution  plays  an  import- 
ant part  in  the  destruction  of  micro-organisms.  They  found 
that  double  metallic  salts,  in  which  the  metal  is  a  constituent 
of  a  complex  ion  in  which  the  concentration  of  the  dissociated 
metal  ions  is  consequently  very  low,  have  very  little  germicidal 
power,  but  that  simple  salts,  in  which  the  condition  is  reversed, 
have  correspondingly  higher  germicidal  power.  Dissocia- 
tion, therefore,  seems  to  have  much  to  do  with  the  matter. 

Inorganic  Disinfectants. 

ACIDS. — These  agents  are  seldom  employed,  since  the  concentration 

required  makes  them  objectionable. 

ALKALIS. — The  same  holds  good  with  regard  to  these  agents. 
SALTS. — In  this  group  we  find  some  of  the  most  powerful  and  most 

useful  germicidal  substances. 

Copper  Sulfate. — It  is  curious  and  interesting  that  while  this 
salt  is  highly  destructive  to  algae  and  other  low  forms  of 
vegetable  life,  it  is  not  of  much  value  for  the  destruction  of 
bacteria.  Its  chief  use  is  for  the  destruction  of  the  green  algae 
that  sometimes  render  the  water  of  reservoirs  dirty  and  offen- 
sive. Some  of  the  salt  contained  in  a  gunny-sack  and  per- 
mitted to  drag  to  and  fro  over  the  surface  of  the  water  behind 
a  slowly  rowed  boat  usually  accomplishes  the  end,  the  actual 
quantity  dissolving  in  the  water  being  almost  infinitesimal. 
Mercuric  Chlorid  (HgCl2). — This  is  probably  the  most  generally 

useful  as  well  as  one  of  the  strongest  germicides. 

A  study  of  its  activity  under  varying  conditions  is  instructive  as  ex- 
emplifying the  varying  behavior  of  germicides  under  the  varying  con- 
ditions under  which  they  may  be  employed. 

First,  it  makes  great  difference  whether  the  mercuric  chlorid  is  added 
to  the  substratum  containing  the  bacteria,  or  whether  the  bacteria  are 
added  to  solutions  of  the  germicide. 

Thus,  when  the  salt  is  dissolved  in  gelatin  in  a  concentration  of 
i:  i,ooo..ooo,  anthrax  bacilli  cannot  grow.  If  it  is  dissolved  in  blood- 
serum,  the  concentration  must  be  increased  to  i :  10,000  to  prevent  their 
growth. 

When  the  anthrax  spores  are  dropped  in  solutions  of  the  salt,  Kronig 
and  Paul  found  that  they  were  killed  in  twelve  to  fourteen  minutes  by 
i :  65  solutions;  in  eighty  minutes  by  i :  500  solutions,  and  in  two  hours 
by  i :  looo  solutions.  When  the  reaction  takes  place  in  albuminous 
media  Behring  and  Nochtf  found  that  much  more  time  was  required. 
Thus,  the  destruction  of  the  spores  by  a  i :  100  solution  required  eighty 
minutes,  and  a  i :  1000  solution  twenty-four  hours  to  completely  kill  all 
of  the  spores. 

LaplaceJ  and  Panfili§  found  that  the  addition  of  5  per  cent,  of  tar- 
taric  or  hydrochloric  acid  facilitated  the  germicidal  action  through  the 

*  "Zeitschrift  fur  Hygiene,"  1897,  xxv,  i.  t  Ibid.,  ix,  432. 

J  "Deutsche  med.  Wochenschrift,"  1887,  866;  1888,  121. 
§  "Ann.  Ig.  Roma,"  1893,  m,  527. 


214  Sterilization  and  Disinfection 

prevention  of  albuminate  of  mercury  formation.  Liibbert  and  Schneider 
and  Behring  have  used  sodium  chlorid  and  ammonium  chlorid.  Both 
of  these  salts  diminish  the  germicidal  action  of  the  mercuric  salt  about 
one-half.  Notwithstanding  this,  however,  the  "antiseptic  tablets"  in 
common  use  for  surgical  and  household  purposes  contain  one  or  both 
of  these  salts,  added  for  the  purpose  of  preventing  the  precipitation  of 
the  mercuric  compounds  formed  in  the  presence  of  alkaline  albuminous 
materials,  such  as  blood,  pus,  sputum,  feces,  etc. 

The  addition  of  about  25  per  cent,  of  alcohol  to  the  solution  of  the 
mercuric  salt  greatly  enhances  its  value.  Strong  alcoholic  solutions 
are,  however,  less  useful  than  aqueous  solutions,  for  the  95  or  100  per 
cent,  alcohol  dehydrates  the  micro-organisms  and  prevents  the  diffusion 
currents  by  which  the  mercury  is  carried  into  their  substance. 

For  most  purposes  a  i :  2000  solution  of  the  mercuric  chlorid  is  to  be 
recommended. 

Silver  Nitrate  (AgNO3). — The  solutions  of  this  salt  are  probably 
more  useful  than  the  frequency  of  their  employment  might  sug- 
gest. They  have,  however,  the  disadvantages  of  decomposing 
when  kept  in  the  light  and  of  making  black  stains  when  applied 
in  concentrated  form  to  the  skin  or  dressings. 

The  germicidal  power  of  the  salt  in  aqueous  solution  is  less  than 
that  of  the  mercuric  chlorid,  but  the  power  in  albuminous  fluids 
is  greater.  Anthrax  spores  in  blood-serum  are  killed  in  seventy 
hours  in  a  i:  12,000  solution.  The  addition  of  other  salts,  as 
ammonium  salts,  interferes  with  the  germicidal  activity  by  in- 
hibiting ionization. 

Combinations  of  the  silver  nitrate  with  albuminous  compounds, 
and  variously  known  as  argonin,  argentum  casein,  argyrol,  pro- 
targol,  etc.,  have  been  used  where  the  disinfecting  power  of  the 
silver  is  sought  for  with  the  least  amount  of  irritation  and  the 
deepest  degree  of  penetration,  as  in  the  treatment  of  gonorrhea. 
Potassium  Permanganate  (KMnOJ. — Solutions  of  this  salt  seem  to 
act  by  virtue  of  a  strong  oxidizing  power.  In  2  per  cent,  solu- 
tions anthrax  spores  are  killed  in  forty  minutes;  in  4  per  cent, 
solutions,  within  fifteen  minutes.  Koch's  experiments  showed 
less  activity  of  the  germicidal  power  against  anthrax  spores. 
In  his  hands  a  5  per  cent,  solution  seemed  to  require  about  a  day 
to  effect  complete  distraction.  A  i  per  cent,  solution  kills  the 
pus  cocci  in  ten  minutes;  a  i :  10,000  solution  kills  plague  bacilli 
in  five  minutes. 

The  chief  difficulty  in  the  way  of  successfully  employing  this 
salt  is  that  it  is  quickly  reduced  and  its  strength  destroyed  by  the 
organic  substrata  in  which  the  bacteria  are  contained. 
HALOGENS  AND  COMPOUNDS. — Those  with  the  lowest  atomic  weight 

have  the  greatest  disinfecting  power. 

Chlorin. — This  is  usually  employed  in  the  form  of  chlorinated 
lime.  It  seems  to  be  a  mixture  of  calcium  hypochlorite,  Ca(ClO2), 
and  calcium  chlorid,  CaOCl2.  The  addition  of  any  acid,  in- 
cluding the  atmospheric  CO2,  causes  the  evolution  of  Cl.  The 
powder  is  readily  soluble  and  solutions  of  i :  500  kill  vegetative 
forms  of  most  bacteria  in  a  few  minutes  (not,  however,  resisting 
spores). 

A  proprietary  compound  known  as  "electrozone,"  made  by 
electrolyzing  sea-water  in  such  a  manner  that  magnesia  and 
chlorin  are  liberated  and  magnesium  hypochlorite  and  magne- 
sium chlorid  formed,  is  a  cheap  and  useful  chlorin  disinfectant. 
Nissen  found  that  1.5  per  cent,  of  it  killed  typhoid  bacilli  in  a 
few  minutes;  Rideal,  that  i  :  400  to  500  dilutions  of  it  disinfected 
sewage  in  fifteen  minutes;  and  Delepine,  that  i  :  50  (equal  to 


Organic  Disinfectants  215 

0.66  per  cent,  of  chlorin)  rapidly  killed  the  tubercle  bacillus 
and  i :  10  (equal  to  3.3  per  cent,  chlorin)  killed  anthrax  spores. 
lodin  Terchlorid  (IC13). — This  compound,  which  is  so  unstable 
that  it  only  keeps  in  an  atmosphere  of  Cl-gas,  has  great  germi- 
cidal action,  that  probably  depends  upon  the  readiness  with  which 
it  decomposes.  In  solutions  of  i :  1000  it  kills  vegetative  bac- 
teria in  a  few  minutes,  and  in  i :  100  it  kills  anthrax  spores  with 
equal  rapidity.  The  presence  of  organic  and  albuminous  mate- 
rials does  not  interfere  with  the  germicidal  action. 

Organic  Disinfectants. 

Carbolic  acid  (C6H5OH)  is  the  most  important  and  generally  useful 
of  these.  It  has  the  advantage  of  being  cheap  and  easily  kept 
and  handled.  In  the  pure  state  it  consists  of  colorless  acicular 
crystals.  When  exposed  to  the  atmosphere  it  takes  up  water 
and  gradually  becomes  a  brownish-yellow  oily  fluid.  The 
crystals  and  deliquesced  crystals  have  powerful  escharotic  proper- 
ties and  cannot  be  touched  without  destruction  of  the  skin. 
In  2  to  3  or  5  per  cent,  solutions  carbolic  acid  destroys  most 
bacteria  within  a  few  minutes.  Anthrax  and  other  powerfully 
resisting  spores,  however,  require  prolonged  exposure.  Tetanus 
spores  are  said  not  to  be  killed  in  less  than  fifteen  hours.  There 
is  no  ionization ;  the  reagent  seems  to  act  by  coagulating  the  bac- 
terial protoplasm. 

Though  carbolic  acid  has  been  for  a  quarter  of  a  century  a 
favorite  surgical  disinfectant,  the  application  of  5  per  cent, 
solution  to  the  skin  has  so  frequently  caused  gangrene  that  it  is 
at  present  in  some  merited  disfavor. 

Closely  related  to  carbolic  acid  and  other  products  of  coal-tar 
distillation  are  orthocresol,  metacresol,  and  paracresol.  "  Tri- 
kresol,"  a  much  used  antiseptic,  is  a  commercial  product  con- 
sisting of  a  mixture  of  all  three  of  the  cresols.  It  is  more  strongly 
germicidal  than  carbolic  acid,  but  is  less  soluble  in  water.  It  is 
or  has  been  largely  used  for  addition  to  therapeutic  serums  in 
the  proportion  of  0.4  per  cent,  as  an  antiseptic.  Such  addition 
causes  the  formation  of  an  albuminous  precipitate  in  which, 
doubtless,  much  of  the  antiseptic  is  lost,  for  upon  its  removal  or 
even  upon  its  sedimentation  resisting  forms  of  bacteria  may 
grow  in  the  serum.  It  cannot,  therefore,  be  looked  upon  as  a 
reliable  preservative. 

"Lysol"  is  said  to  be  a  solution  of  coal-tar  cresol  in  potassium 
soap.  It  has  the  advantage  of  forming  a  lather-like  soap,  so 
that  it  can  be  employed  both  as  a  cleanser  and  disinfectant.  In 
i  per  cent,  solutions  it  is  capable  of  destroying  cocci,  typhoid 
bacilli,  and  other  micro-organisms  of  low  resisting  power. 

"Creolin"  is  also  a  combination  of  cresols  with  potassium 
soap.  When  added  to  water  it  immediately  forms  an  emulsion. 
It  has  been  much  used  in  obstetric  practice,  where  it  has  earned 
more  reputation  than  it  deserves. 

''Formalin." — This  is  Schering's  commercial  denomination  of  a 
30  to  40  per  cent,  aqueous  solution  of  formaldehyd  gas  (H — COH) 
or  formic  aldehyd.  The  solution  is  highly  germicidal  so 
long  as  it  is  fresh.  When  exposed  for  long  to  the  atmosphere 
it  polymerizes  into  trioxymethylene  and  paraformaldehyde 
and  greatly  loses  its  power.  A  10  per  cent,  solution  of  formalin 
kills  pus  cocci  in  half  an  hour.  A  5  per  cent,  solution  kills  cholera 
spirilli  in  three  minutes;  anthrax  bacilli,  in  fifteen  minutes; 
anthrax  spores,  in  five  hours.  Pure  formalin  kills  anthrax  spores 


2l6 


Sterilization  and  Disinfection 


in  ten  to  thirty  minutes.  Strong  solutions  are  extremely  irri- 
tating and  so  not  applicable  in  surgery.  They  are,  however,  of 
great  use  for  household  disinfection.  Formalin  and  formaldehyd 
gas  find  their  chief  usefulness  for  the  aerial  disinfection  of  sick 
chambers  and  domiciles,  where  they  are  either  used  as  a  spray 
or  the  gas  evolved  by  chemical  means  or  by  heat,  as  will  be  shown 
below. 

Peroxid  of  hydrogen  (H2O2)  is  germicidal  through  its  power  to 
liberate  the  nascent  O.  It  quickly  decomposes  when  brought 
into  contact  with  organic  matter,  and,  therefore,  has  a  very  limited 
sphere  of  usefulness. 

The  following  tables,  compiled  by  Hiss  from  Fliigge,  will 
show  the  comparative  values  of  the  commonly  employed 
antiseptics  and  germicides: 


INHIBITION  STRENGTHS  OF  VARIOUS  ANTISEPTICS. 
(Adapted  from  Fliigge,  Leipzig,  1902.) 


Anthrax 
Bacilli. 

Other  Bacteria. 

Putrefactive 
Bacteria  in 
Bouillon. 

ACIDS 
Sulphuric  
Hydrochloric 

i  :  3000 
i  :  3000 

Choi.  spir.  i  :  6000 

Sulphurous 

B.  mallei  i  :  700 
B.  typh.  i  :  500 

i  :  6000 

Arsenous  

i  :  200 

Boric  

i  :  800 

i  :  100 

ALKALIES 
Potass,  hydrox  

i  :  700 

B.  diph.  i  :  600 

Ammon   hydrox 

i  :  700 

Choi,  spir   i  :  400 
B.  typh.  i  :  400 
Choi,  spir   i  •  500 

Calcium  hydrox. 

B.  typh.  i  :  500 
Choi    spir    i  *  i  ico 

SALTS 
Copper  sulphate. 

B,  typh.  i  :  1  100 

Ferric  sulphate  

Mercuric  chlorid  .  . 

B.  typh.  i  :  60,000 

Silver  nitrate  

i      6o,coo 

Choi,  spir., 

Potass,  perman. 

I       IOOO 

B.  typhosus  i  :  50,000 

HALOGENS  AND  COMPOUNDS 
Chlorin  

i     1500 

Bromin 

lodin  

i     5000 

Potass,  iodid 

Sodium  chlor  
ORGANIC  COMPOUNDS 
Ethyl  alcohol  

i    60 

I       12 

I       IO 

Acetic  and  oxalic  acids  

B.  diph.  i  :  500 

Carbolic  acid 

I       8oO 

B  typh.  i  •  400 

Benzoic  acid 

I        IOOO 

Choi.  spir.  i  :  600 

Salicylic  acid  
Formalin  (40%  lormaldehyd) 

I     1500 

Choi.  spir.  i  :  20,000 

Camphor  

i       1000 

Staphylo.  i  :  5000 

Thymol 

Oil  mentha  pip  

I        3000 

Oil  of  terebinth  

I      8000 

Peroxid  of  hydrogen 

Comparison  of  Disinfectants 


217 


BACTERICIDAL  STRENGTH  OF  COMMON  DISINFECTANTS. 
(Adapted  from  Fliigge,  Leipzig,  1902.) 


Streptococci 
and  Sta- 
phylococci. 

Anthrax  and  Typhoid  Bacilli, 
Cholera  Spirillum. 

Anthrax  spores. 

5  minutes. 

5  minutes. 

2  to  24  hours. 

ACIDS 
Sulphuric  

i  :  10 
i  :  10 

i  :  100 
i  :  100 

i  :  1500 
i  :  1500 
Typhoid  i  :  700 
i  :  300  (Gas  10 
vol.  %) 
i  =3° 

i  :  50  in  10  days 
i  :  50  in  10  days 

Cone.  sol.  in  com- 
plete disinfection 

i  :  20  (5  days) 
i  :  2000  (26  hrs.) 

i  :  20  (i  day) 
i  :  20  (i  hour) 

2%  (in  i  hour) 
i  :  iooo(in  12  hrs.) 

Alcol.  50%  for  4 
months     with- 
out   killing1 
spores  (Koch*) 
i    :  20   (4  to  45 
days)    (at    40° 
in  3  hours) 

(10%  in  5  hours) 

i  :  20  (in  6  hrs.) 
i  :  100  (in  i  hr.) 
3  :  loo  (in  i  hr.) 

Hydrochloric 

Sulphurous  

Sulphurous    .               ... 

Boric  

ALKALIES 
Potass,  hydrox. 

i  :  5 

i  :3oo 
i  :  300 
i  :  looo 

Ammon   hydrox 

Calcium  

SALTS 
Copper  sulphate  

Mercuric  chlor.     .    . 

i  :  10,000  to 

IOOO 

i  :  2coo 

i  :  10,000 
i  :  4000 

Potass,  permang' 

i  :  200 

"Calc.  chlorid."  

i  :  500 

i% 
i  :  looo 

70%  —  10 
minutes 

Cholera  i  :  200 
Typh.  i  :  50 

i  :  300 
i  :  100 

i  :  20 
i  :  200 

HALOGENS  AND  COMPOUNDS 

i% 
i  :  200 

70%  —  15 
minutes 

i  :6o 

i  :  300 

Trichloridof  iodin  
ORGANIC  COMPOUNDS 
Ethyl  alcohol  

i  :  200  to  300 
i  -.300 

i  :  3000 

i  :  looo 
i  :  500 

Acetic  and  oxalic  acids. 
Carbolic  acid  

Lysol  

Creolin 

Salicylic  acid  

i  :  1000 
i  :  TO 
Cone. 

Formalin  (40%  formaldehyd) 
Peroxid  of  hydrogen  

Certain  fundamental  principles  govern  the  rationale  of 
disinfection,  and  must  be  kept  in  mind :  (i)  the  reagent  em- 
ployed should  be  known  to  act  destructively  upon  bacteria ; 
(2)  it  must  be  applied  to  the  bacteria  to  be  killed ;  (3)  it  must 
be  applied  in  sufficiently  concentrated  form,  and  (4)  it  must 
be  left  in  contact  with  the  bacteria  long  enough  to  accom- 
plish the  effect  desired. 

During  the  period  of  illness  the  chamber  in  which  the 
patient  is  confined  should  be  freely  ventilated.  An  abun- 
dance of  fresh,  pure  air  is  a  comfort  to  the  patient  and  a 
protection  to  the  doctor  and  nurse. 

After  recovery  or  death  one  should  rely  less  upon  fumi- 
gation than  upon  disinfection  of  the  walls  and  floor,  the 

*  Koch,  Arb.  a.  d.  kais.  Gesundheitsamt,  i,  1881. 


218  Sterilization  and  Disinfection 

similar  disinfection  of  the  wooden  part  of  the  furniture,  and 
the  sterilization  of  all  else.  The  fumes  of  sulphur  do  some 
good,  especially  when  combined  with  steam,  but  are  greatly 
overestimated  in  action  and  are  very  destructive  to  furnishings, 
so  that  they  are  rapidly  giving  way  to  the  more  satisfactory, 
less  destructive,  and  equally  germicidal  formaldehyd  vapor. 

Formaldehyd  is  probably  the  best  germicide  that  has  yet 
been  recommended.  Its  use  for  the  disinfection  of  rooms 
and  hospital  wards  was  first  suggested  by  Trillat*  in  1892,  but 
it  did  not  make  much  stir  in  the  medical  world  until  a  year 
or  more  had  passed  and  a  40  per  cent,  solution  of  the  gas, 
under  the  name  of  "  Formalin,"  had  been  placed  upon  the 
market.  Care  must  be  exercised  in  handling  the  fluid, 
that  the  hands  do  not  become  wet  with  it,  as  it  hardens  the 
skin  and  deadens  sensation.  The  vapor  is  exceedingly  irri- 
tating to  the  mucous  membrane  of  the  eyes  and  nose. 

The  solution  can  be  employed  to  spray  the  walls  and  floors 
of  rooms,  though  Rosenauf  finds  that  unless  the  spray  dis- 
charged from  a  large  atomizer  be  very  fine,  its  action  is  un- 
certain. 

The  original  method  of  disinfection,  suggested  by  Robin- 
son,! consisted  of  the  evolution  of  the  gas  by  volatilizing 
methyl  alcohol,  and  passing  the  vapor  over  heated  asbestos. 
Shortly  many  efficient  forms  of  apparatus  were  placed  upon 
the  market,  for  the  evolution  of  the  gas  or  for  discharging 
it  from  the  solution. 

It  is  not  necessary  to  use  a  special  apparatus  in  order 
to  disinfect  with  formaldehyd;  one  can,  in  an  emergency, 
hang  up  a  number  of  sheets,  saturated  with  the  40  per  cent, 
solution,  in  the  room  to  be  disinfected. 

The  number  of  sheets  must  vary  with  the  size  of  the 
room,  as  each  is  able  to  evolve  but  a  certain  amount  of  the 
gas,  and  the  quantity  necessary  for  disinfection  varies  with 
the  size  of  the  room. 

A  better  method  of  evolving  the  gas  for  purposes  of  dis- 
infection devised  by  Evans  and  Russell  §  is  to  combine  the 
solution  with  permanganate  of  potassium,  when  an  almost 
explosive  liberation  of  the  gas  takes  place. 

*  "Compte  rendu  de  1'Acad.  des  Sciences,"  Paris,  1892. 
t  "Disinfection  and  Disinfectants,"  P.  Blakiston's  Son  &  Co.,  Phila- 
delphia, 1902. 

}  "Ninth  Report  of  the  State  Board  of  Health  of  Maine,"  1896. 
§  "Report  of  the  State  Board  of  Health  of  Maine,"  1904. 


Disinfection  of  Sick-chambers,  etc.  219 

Frankforter*  found  that  a  good  method  of  escaping  the 
undesirable  features  of  the  gaseous  evolution  was  to  mix  the 
powder  of  permanganate  of  potassium  with  an  equal  volume 
of  sand,  so  that-  the  formaldehyd  solution  is  brought  more 
slowly  into  contact  with  the  permanganate,  under  condi- 
tions unfavorable  to  the  formation  of  oxids  of  manganese, 
such  as  otherwise  tend  to  coat  the  grains  of  permanganate 
and  prevent  further  reaction  between  the  formaldehyd  solu- 
tion and  the  permanganate. 

The  employment  of  calcium  carbide  for  the  same  purpose 
is  suggested  by  Evans. f  The  best  results  were  obtained 
when  the  calcium  carbide  was  in  lumps  about  the  size  of  a 
pea;  when  the  formaldehyd  solution  was  diluted  with  an 
equal  volume  of  water,  and  when  the  diluted  formaldehyde 
was  added  to  the  carbide  in  the  proportion  of  5  c.c.  of  the 
former  to  3  grams  of  the  latter.  In  the  permanganate 
method  the  quantity  of  formalin  (or  37-40  per  cent,  for- 
maldehyd in  water)  should  equal  200  c.c.  to  1000  cubic  feet 
of  space,  but  in  the  carbide  method  500  c.c.  must  be  used  to 
achieve  the  same  result.  Evans,  therefore,  prefers  the 
permanganate  method. 

To  disinfect  with  formaldehyd  or  any  gaseous  disinfectant, 
the  room  must  be  carefully  closed,  the  cracks  of  the  windows 
and  doors  being  sealed  by  pasting  strips  of  paper  over  them. 
If  an  apparatus  is  used,  it  is  set  in  action,  the  discharged 
vapor  entering  the  room  through  the  keyhole  or  some  other 
convenient  aperture,  the  gas  being  allowed  to  act  undis- 
turbed for  some  hours,  after  which  the  windows  and  doors 
are  all  thrown  open  to  fresh  air  and  sunlight. 

If  sheets  are  hung  up,  or  the  permanganate  method 
employed,  the  windows  and  doors,  other  than  that  by  means 
of  which  the  operator  is  to  escape,  are  closed  and  sealed. 
A  dish-pan  or  wash-tub  is  placed  in  the  center  of  the  room, 
and  in  it  the  can  containing  the  permanganate  and  sand. 
The  formaldehyd  solution  is  poured  on,  the  operator  making 
his  escape,  closing  and  sealing  the  door  behind  him.  Any 
closets  in  the  room  must  be  left  open  so  that  they  and  their 
contents  may  be  disinfected  with  the  room. 

So  far  as  is  known  at  present,  superficial  disinfection  by 
formaldehyd  leaves  little  to  be  desired.  Care  must,  how- 

*  ''Reports  and  Papers  of  the  American  Public  Health  Association," 
vol.  xxxii,  part  n,  p.  114,  1906. 
t  Ibid.,  p.  108. 


220  Sterilization  and  Disinfection 

ever,  be  exercised  to  see  that  the  required  volume  of  gas 
is  generated  to  disinfect  the  apartment.  A  sufficient  con- 
centration of  the  gas  is  absolutely  necessary,  and  the  method 
selected  should  be  one  capable  of  discharging  the  gas  in  a 
short  time,  so  that  it  immediately  pervades  the  atmosphere. 

Disinfection  with  formaldehyd  is,  however,  only  super- 
ficial, its  penetrating  powers  being  limited.  The  discharge 
of  gas  into  the  room  should  only  be  preliminary  to  other  and 
more  thorough  disinfection  and  sterilization  of  the  contents 
by  the  application  of  solutions  of  disinfectants  to  the  wood- 
work, and  to  the  boiling  of  the  linen,  etc. 

The  Dejecta.— In  diphtheria  the  expectoration  and  nasal 
discharges  are  highly  infectious  and  should  be  received  in  old 
rags  or  in  Japanese  paper  napkins — not  handkerchiefs  or 


Fig.  43. — Pasteboard  cup  for  receiving  infectious  sputum.     When  used 
the  pasteboard  can  be  removed  from  the  iron  frame  and  burned. 

towels — and  should  be  burned.  The  sputum  of  tuberculous 
patients  should  either  be  collected  in  a  glazed  earthen  vessel 
which  can  be  subjected  to  boiling  and  disinfection,  or,  as  is 
an  excellent  plan,  should  be  received  in  Japanese  rice-paper 
napkins,  which  can  at  once  be  burned.  These  napkins  are 
not  quite  so  good  as  the  small  pasteboard  boxes  (Fig.  43) 
recommended  by  some  city  boards  of  health,  because, 
being  highly  absorbent,  the  sputum  is  apt  to  soak  through 
and  soil  the  ringers.  For  the  fastidious  patients  in  cer- 
tain sanatoria,  cut-glass  bottles  with  tightly  fitting  lids 
are  used  to  collect  the  sputum,  and  as  these  are  not  un- 
sightly, the  patients  make  no  objection  to  carrying  them 
about  with  them.  Tuberculous  patients  should  be  provided 
with  rice-paper  instead  of  handkerchiefs,  and  should  have 
their  towels,  knives,  forks,  spoons,  plates,  etc.,  kept  strictly 
apart  from  the  others  of  the  household  and  carefully  sterilized 
after  using.  Patients  whose  mental  acuity  makes  their 


Disinfection  of  the  Clothing  221 

sensibilities  very  pronounced  need  never  be  told  of  these 
arrangements. 

The  excreta  from  cases  of  typhoid  fever  and  cholera  re- 
quire particular  attention.  These,  and  indeed  all  alvine 
matter  the  possible  source  of  infection  or  contagion,  should 
be  received  in  glazed  earthen  vessels  and  immediately  and 
intimately  mixed  with  a  5  per  cent,  solution  of  chlorinated 
lime  (containing  25  per  cent,  of  chlorin)  if  semi-solid,  or  with 
the  powder  if  liquid,  and  allowed  to  stand  for  an  hour  before 
being  thrown  into  the  drain. 

Thoughtful  consideration  should  always  be  given  the 
germicides  used  to  disinfect  the  discharges,  lest  combination 
of  the  chemical  with  ingredients  of  the  discharge  produce 
inert  compounds.  Thus,  bichlorid  of  mercury  cannot  be 
used  because  it  forms  an  inert  compound  with  albumin. 

The  Clothing,  etc. — The  bed-clothing,  towels,  napkins, 
handkerchiefs,  night-robes,  underclothes,  etc.,  used  by  a 
patient  suffering  from  an  infectious  disease,  as  well  as  the 
towels,  napkins,  handkerchiefs,  caps,  aprons,  and  outside 
dresses  worn  by  the  nurse,  should  be  regarded  as  infective 
and  carefully  sterilized.  The  only  satisfactory  method  of 
doing  this  is  by  prolonged  subjection  to  steam  in  a  special 
apparatus;  but,  as  this  is  only  possible  in  hospitals,  the 
next  best  thing  is  boiling  for  some  time  in  the  ordinary 
wash-boiler.  In  drying,  the  wash  should  hang  longer  than 
usual  in  the  sun  and  wind.  Woolen  underwear  can  be 
treated  exactly  as  if  made  of  cotton.  The  woolen  outer 
clothing  of  the  patient,  if  infective,  requires  special  treat- 
ment. Fortunately,  the  infection  of  the  outer  garments 
is  unusual.  The  only  reliable  method  for  their  sterilization 
is  prolonged  exposure  to  hot  air  at  1 10°  C.  In  private  prac- 
tice it  often  becomes  a  grave  question  what  shall  be  done 
with  these  articles.  Prolonged  exposure  to  fresh  air  and 
sunlight  will,  however,  aid  in  rendering  them  harmless; 
and  can  be  practised  when  it  is  not  certain  that  they  are 
actually  infective.  Infective  articles  of  wool  may  be  sent  to 
the  city  hospital  or  to  one  of  the  moth-destroying  and  fumi- 
gating establishments  which  can  be  found  in  all  large  cities, 
and  baked. 

The  doctor  visiting  a  case  of  dangerous  infection  or  a  hos- 
pital for  infectious  diseases  should  cover  his  clothing  with  a 
linen  or  cotton  gown,  and  protect  his  hair  with  a  cap,  these 
articles  being  disinfected  after  the  visit.  By  such  precau- 


222  Sterilization  and  Disinfection 

tions  he  will  avoid  spreading  infection  among  his  patients  or 
carrying  it  to  his  own  family. 

The  Furniture,  etc. — The  destruction  of  infective  fur- 
niture is  unnecessary.  The  doctor  treating  a  case  of  infec- 
tious disease,  if  he  properly  perform  his' functions,  will  save 
much  trouble  and  money  for  his  patient  by  ordering  his 
immediate  isolation  in  an  uncarpeted,  scantily,  and  simply 
furnished  room  the  moment  an  infectious  disease  is  sus- 
pected. However,  if  before  his  removal  the  patient  has 
occupied  another  bed,  its  clothing  should  be  promptly  dis- 
infected. 

After  the  recovery  or  death  of  the  patient  the  walls  and 
ceiling  of  the  room  should  be  sprayed  with  a  formaldehyd 
solution,  or  the  room  sealed  and  filled  with  the  vapor. 
If  they  are  hung  with  paper,  they  should  be  dampened  with 
i :  1000  bichlorid  of  mercury  solution  before  new  paper  is 
hung. 

Strehl  has  demonstrated  that  when  10  per  cent,  formalin 
solution  is  sponged  upon  artificially  infected  curtains,  etc., 
the  bacteria  are  killed  by  the  action  of  the  disinfectant. 
This  is  an  important  adjunct  to  our  means  of  disinfecting 
the  furniture  of  the  sick-chamber. 

The  floor  should  be  scoured  with  40  per  cent,  formaldehyd 
solution,  5  per  cent,  carbolic  acid  solution,  or  i  :  1000  bi- 
chlorid of  mercury  solution  (no  soap  being  used,  as  it 
destroys  the  bichlorid  of  mercury  and  prevents  its  action), 
and  all  the  wooden  articles  wiped  off  two  or  three  times 
with  one  of  the  same  solutions.  If  a  straw  mattress  was  used 
it  should  be  burned  and  the  cover  boiled.  If  a  hair  mattress 
was  used,  it  can  be  steamed  or  baked  by  the  manufacturers, 
who  usually  have  ovens  for  the  purpose  of  destroying  moths, 
but  which  answer  for  sterilizing  closets.  Curtains,  shades, 
etc.,  should  receive  proper  attention;  but,  of  course,  the 
greater  the  precautions  exercised  in  the  beginning,  the  fewer 
the  articles  that  will  need  attention  in  the  end. 

The  Patient,  whether  he  live  or  die,  may  be  a  means 
of  spreading  the  disease  unless  specially  cared  for.  After 
convalescence  the  body  should  be  scoured  with  biniodide  of 
mercury  soap,  bathed  with  a  weak  bichlorid  of  mercury 
solution  or  with  a  2  per  cent,  carbolic  acid  solution,  or  with 
25-50  per  cent,  alcohol,  before  the  patient  is  allowed  to 
mingle  with  society,  and  the  hair  should  either  be  cut  off 
or  carefully  washed  with  the  disinfecting  solution  or  an 


Disinfection  of  the  Patient  223 

antiseptic  soap.  In  desquamative  diseases  it  seems  best 
to  have  the  entire  body  anointed  with  cosmolin  once  daily, 
beginning  before  desquamation  begins  and  having  the 
unguent  well  rubbed  in,  in  order  to  prevent  the  particles  of 
epidermis,  in  which  the  specific  contagium  probably  occurs, 
being  distributed  through  the  atmosphere.  Carbolated 
may  be  better  than  plain  cosmolin,  not  because  of  the  very 
slight  antiseptic  value  it  possesses,  but  because  it  helps  to 
allay  the  itching  which  may  accompany  the  desquamative 
process. 

After  the  patient  is  about  the  room  again,  common  sense 
will  prohibit  the  admission  of  visitors  until  the  suggested 
disinfective  measures  have  been  adopted,  and  after  this, 
touching,  and  especially  kissing  him,  should  be  avoided  for 
some  time. 

The  bodies  of  those  that  die  of  infectious  diseases  should  be 
washed  in  a  strong  disinfectant  solution,  and  should  be  given 
a  strictly  private  funeral.  If  this  be  impossible,  the  body 
should  be  sealed  in  the  coffin  and  only  the  face  viewed 
through  a  plate  of  glass.  In  my  judgment,  the  body  is 
best  disposed  of  by  cremation. 

A  dead  body  cannot  remain  a  source  of  infection  for  an 
indefinite  period.  Esmarch,*  who  made  a  series  of  labora- 
tory experiments  to  determine  the  fate  of  pathogenic  bac- 
teria in  the  dead  body,  found  that  in  septicemia,  cholera, 
anthrax,  malignant  edema,  tuberculosis,  tetanus,  and  typhoid 
fever  the  pathogenic  bacteria  all  die  sooner  or  later,  more 
rapidly  during  active  decomposition  than  during  preservation 
of  the  tissues. 

*  "Zeitschrift  fur  Hygiene,"  1893. 


CHAPTER   VII. 

CULTURE-MEDIA   AND   THE  CULTIVATION  OF 
MICRO-ORGANISMS. 

IN  order  to  observe  them  accurately  the  organisms  must  be 
separated  from  their  natural  surroundings  and  artificially 
cultivated  upon  certain  prepared  media  of  standard  compo- 
sition, in  such  a  manner  that  only  organisms  of  the  same 
kind  are  together.  The  effects  of  one  organism  upon  the 
growth  of  another,  by  neutralizing  its  metabolic  products, 
by  changing  the  reaction  of  the  medium  in  which  it  grows  so 
as  to  inhibit  further  multiplication,  by  dissolving  the  other 
species  through  its  enzymes,  etc.,  suffice  to  show  how  im- 
possible it  is  to  determine  the  natural  history  of  any  organ- 
ism unless  it  be  kept  strictly  away  from  other  species. 

Fortunately  the  same  general  principles  apply  equally  for 
the  cultivation  of  all  forms  of  micro-organismal  life,  and  much 
the  same  media  apply  in  all  cases.  What  is  said,  therefore, 
about  the  bacteria  may  be  regarded  as  appropriate  for  all. 

Various  organic  and  inorganic  mixtures  have  been  sug- 
gested for  the  cultivation  of  bacteria,  but  few  have  met  with 
particular  favor  and  become  standards.  At  the  present 
time  certain  standard  media  are  used  in  every  laboratory 
in  the  world;  all  systematic  study  of  the  organisms  depends 
upon  the  behavior  of  bacteria  upon  them,  and  no  study 
of  micro-organisms  can  be  regarded  as  complete  unless  the 
behavior  of  the  bacteria  upon  them  has  been  carefully 
considered. 

Our  studies  of  the  biology  of  the  bacteria  have  shown  that 
they  grow  best  in  mixtures  containing  at  least  80  per  cent,  of 
water,  of  neutral  or  feebly  alkaline  reaction,  and  of  a  com- 
position which,  for  the  pathogenic  forms  at  least,  should 
approximate  the  juices  of  the  animal  body.  It  might  be 
added  that  transparency  is  a  very  desirable  quality,  and  that 
the  most  generally  useful  culture  media  are  those  that  can 
be  liquefied  and  solidified  at  will. 

224 


Cultivation  of  Micro-organisms  225 

All  accurate  bacteriologic  culture  experiments  require 
that  an  exact  knowledge  of  the  chemistry  of  the  media  used 
shall  be  at  hand.  The  importance  of  this  and  the  necessity 
for  having  exact  information  regarding  the  reaction  of  the 
media  are  well  brought  out  in  the  following  excerpts  from 


Fig.  44. — Buret  for  titrating  media.     (From  Hiss  and  Zinsser,  "Text- 
Book  of  Bacteriology,"  D.  Appleton  &  Co,,  Publishers.) 

the  Report  of  the  Committee  of  Bacteriologists  of  the  Amer- 
ican Public  Health  Association  :* 

"The  first  thing  to  obtain  is  a  standard  'indicator'  which  will  give 
uniform  results.  These  requirements  are  best  fulfilled  by  phenol- 
phthalein." 

"  The  question  of  the  proper  reaction  of  media  for  the  cultivation 
of  bacteria  and  the  method  of  obtaining  this  reaction  have  been  dis- 
cussed in  a  valuable  paper  by  Mr.  George  W.  Fuller,  published  in  the 

*  "Jour.  Amer.  Public  Health  Assoc.,"  Jan.,  1898,  p.  72. 
15 


226  Cultivation  of  Micro-organisms 

'Journal  of  the  American  Public  Health  Association,'  Oct.,  1895, 
vol.  xx,  p.  321." 

"Method  of  determining  the  degree  of  reaction  of  culture  media  : 
For  this  most  important  part  in  the  preparation  of  culture  media, 
burets  graduated  into  one-tenth  c.c.  and  three  solutions  are  required — 

"1.  A  0.5  per  cent,  solution  of  commercial  phenolphthalein,  in  50 
per  cent,  alcohol. 

"2.  A  —  solution  of  sodium  hydroxid. 

"3.  A  —  solution  of  hydric  chlorid. 

"Solutions  2  and  3  must  be  accurately  made  and  must  correspond 
with  the  normal  solutions  soon  to  be  referred  to. 

"Solutions  of  sodium  hydroxid  are  prone  to  deterioration  from  the 
absorption  of  carbon  dioxid  and  the  consequent  formation  of  sodium 
carbonate.  To  prevent  as  much  as  possible  this  change,  it  is  well  to 
place  in  the  bottle  containing  the  stock  solution  a  small  amount  of 
calcium  hydroxid,  while  the  air  entering  the  burets  or  the  supply 
bottles  should  be  made  to  pass  through  a  U-tube  containing  caustic 
soda,  to  extract  from  it  the  carbon  dioxid." 

"  The  medium  to  be  tested,  all  ingredients  being  dissolved,  is  brought 
to  the  prescribed  volume  by  the  addition  of  distilled  water  to  replace 
that  lost  by  boiling,  and  after  being  thoroughly  stirred,  5  c.c.  are 
transferred  to  a  6-inch  porcelain  evaporating-dish.  To  this  45  c.c. 
of  distilled  water  are  added  and  the  50  c.c.  of  fluid  are  boiled  for  three 
minutes  over  a  flame.  One  cubic  centimeter  of  the  solution  of  phenol- 
phthalein (No.  1)  is  then  added,  and  by  titration  with  the  required 
reagent  (No.  2  or  No.  3)  the  reaction  is  determined.  In  the  majority  of 

instances  the  reaction  will  be  found  to  be  acid,  so  that  the  —  sodium 

hydroxid  is  the  reagent  most  frequently  required.  This  determination 
should  be  made  not  less  than  three  times  and  the  average  of  the  results 
obtained  taken  as  the  degree  of  the  reaction. 

"One  of  the  most  difficult  things  to  determine  in  this  process  is 
exactly  when  the  neutral  point  is  reached  as  shown  by  the  color  de- 
veloped, and  to  be  able  in  every  instance  to  obtain  the  same  shade 
of  color.  To  aid  in  this  regard,  it  may  be  remarked  that  in  bright 
daylight  the  first  change  that  can  be  seen  on  the  addition  of  alkali  is 
a  very  faint  darkening  of  the  fluid,  which,  on  the  addition  of  more 
alkali,  becomes  a  more  evident  color  and  develops  into  what  might 
be  described  as  an  Italian  pink.  A  still  further  addition  of  alkali 
suddenly  develops  a  clear  and  bright  pink  color,  and  this  is  the  reac- 
tion always  to  be  obtained.  All  titrations  should  be  made  quickly 
and  in  the  hot  solutions  to  avoid  complications  arising  from  the  presence 
of  carbon  dioxid. 

"The  next  step  in  the  process  is  to  add  to  the  bulk  of  the  medium 
the  calculated  amount  of  the  reagent,  either  alkali  or  acid,  as  may  be 
determined.  For  the  purpose  of  neutralization  a  normal  solution  of 
sodium  hydroxid  or  of  hydric  chlorid  is  used,  and  after  being  thor- 
oughly stirred  the  fluid  thus  neutralized  is  again  tested  in  the  same 
manner  as  at  first,  to  insure  the  proper  reaction  of  the  medium  being 
attained.  When  neutralization  is  to  be  effected  by  the  addition  of 
an  alkali,  it  not  infrequently  happens  that  after  the  calculated  amount 
of  normal  solution  of  sodium  hydroxid  has  been  added,  the  second 
test  will  show  that  the  medium  is  acid  to  phenolphthalein,  to  the 
extent  sometimes  of  0.5  to  1  per  cent.  This  discrepancy  is  perhaps 
due  to  side  reactions  which  are  not  understood.  The  reaction  of  the 
medium,  however,  must  be  brought  to  the  desired  point  by  the  further 


Bouillon  227 

addition  of  sodium  hydroxid,  and  the  titrations  and  additions  of 
alkali  must  be  repeated  until  the  medium  has  the  desired  reaction 
(i.  e.,  0.0  per  cent,  to  0.005  per  cent. ;  see  below). 

"After  the  prescribed  period  of  heating,  it  is  frequently  found  that 
the  medium  is  again  slightly  acid,  usually  about  0.5  per  cent.  Without 
correcting  this,  the  fluid  is  to  be  filtered  and  the  calculated  amount  of 
acid  or  alkali  is  to  be  added  to  change  the  reaction  to  the  one  desired. 
A  still  further  change  in  reaction  is  not  infrequently  to  be  observed 
after  sterilization,  the  degree  of  acidity  varying  apparently  with  the 
composition  of  the  media  and  the  degree  and  continuance  of  the  heat." 

"Manner  of  expressing  the  reaction:  Since  at  the  time  the  reaction 
is  first  determined  culture  media  are  more  often  acid  than  alkaline,  it  is 
proposed  that  acid  media  be  designated  by  the  plus  sign  and  alkaline 
media  by  the  minus  sign,  and  that  the  degree  of  acidity  or  alkalinity 
be  noted  in  parts  per  hundred.  Thus,  a  medium  marked  +1.5 
would  indicate  that  the  medium  was  acid,  and  that  1.5  per  cent,  of 

—  sodium  hydroxid  is  required  to  make  it  neutral  to  phenolphthalein ; 
while  — 1.5  would  indicate  that  the  medium  was  alkaline  and  that 
1.5  per  cent,  of  —  acid  must  be  added  to  make  it  neutral  to  the  indicator." 

''Standard  reaction  of  media  (provisional): 

"Experience  seems  to  vary  somewhat  as  to  the  optimum  degree 
of  reaction  which  shall  be  uniformly  adopted  in  the  preparation  of 
standard  culture  media.  To  what  extent  this  is  due  to  variation  in 
natural  conditions  as  compared  with  variations  of  laboratory  procedure 
it  seems  impossible  to  state.  Somewhat  different  degrees  of  reaction 
for  optimum  growth  are  required,  not  only  in  or  upon  the  media  of 
different  composition  and  by  bacteria  of  different  species,  but  also 
by  bacteria  of  the  same  species  when  in  different  stages  of  vitality. 
The  bulk  of  available  evidence  from  both  Europe  and  America  points 
to  a  reaction  of  -f-1.5  as  the  optimum  degree  of  reaction  for  bacterial 
development  in  inoculated  culture  media.  While  this  experience  is 
at  variance  with  that  in  several  of  our  own  laboratories,  it  has  been 
deemed  wisest  to  adopt  -f-1.5  as  the  provisional  standard  reaction  of 
media,  but  with  the  recommendation  that  the  optimum  growth  reaction 
be  always  recorded  with  the  species." 

Many  bacteriologists  regard  a  reaction  of  +1.0  as  a 
more  desirable  standard  and  use  it  exclusively. 


BOUILLON. 

This  is  one  of  the  most  useful  and  most  simple  media. 
It  can  be  prepared  from  meat  or  from  meat  extract,  and  is 
the  basis  of  most  of  the  culture  media.  The  addition  of  10 
per  cent,  of  gelatin  makes  it  " gelatin" ;  that  of  i  per  cent,  of 
agar-agar  makes  it  "agar-agar."  The  preparation  of  these 
media,  however,  requires  special  directions,  which  will  be 
given  below. 

I.  To  Prepare  Bouillon  from  Fresh  Meat. — To  500  grams 
of  finely  chopped  lean,  boneless  beef,  1000  c.c.  of  clean  water 
are  added  and  allowed  to  stand  for  about  twelve  hours  on 


228  Cultivation  of  Micro-organisms 

ice.  At  the  end  of  this  time  the  liquor  is  decanted,  that 
remaining  on  the  meat  expressed  through  a  cloth,  and  then, 
as  the  entire  quantity  is  seldom  regained,  enough  water 
added  to  bring  the  total  amount  up  to  1000  c.c.  This 
liquid  is  called  the  meat-infusion.  To  it  10  grams  of  Witte's 
or  Fairchild's  dried  beef-peptone  and  5  grams  of  sodium 
chlorid  are  added,  and  the  whole  boiled  until  the  albumins 
of  the  meat -infusion  coagulate,  titrated  or  otherwise  cor- 
rected for  acidity,  boiled  again  for  a  short  time,  and  then 
filtered  through  a  fine  filter-paper.  It  should  be  slightly 
yellow  and  perfectly  clear  and  limpid.  Smith,*  referring  to 
bouillon  intended  for  the  culture  of  diphtheria  bacilli  for 
toxin,  says  that  when  the  peptones  are  added  before  boiling 
most  of  them  are  lost,  and  therefore  recommends  that  the 
meat-infusion  be  boiled  and  filtered  and  the  solid  ingredients 
added  and  dissolved  subsequently.  The  reaction,  which  is 
strongly  acid,  is  then  carefully  corrected  by  titration  accord- 
ing to  the  directions  already  given. 

For  rough  work  in  students'  classes  litmus  paper  may 
be  used  as  an  indicator  for  determining  and  correcting  the 
acidity  resulting  from  the  sarcolactic  and  other  acids  in  the 
meat-infusion,  the  alkaline  solution  being  added  drop  by 
drop  until  a  faint  blue  appears  on  the  red  paper;  or  the 
method  of  using  phenolphthalein  can  be  employed,  the 
addition  of  the  alkaline  solution  being  continued  until  a  drop 
of  the  bouillon  produces  a  red  spot  upon  phenolphthalein 
paper,  made,  as  suggested  by  Timpe,  by  saturating  bibulous 
paper  cut  into  strips  with  a  solution  of  5  grams  of  phenol- 
phthalein to  i  liter  of  50  per  cent,  alcohol.  Acids  do  not 
change  the  appearance  of  the  paper,  but  small  traces  of  alkali 
turn  it  red. 

If  the  bouillon  is  to  be  employed  for  exact  work,  these 
crude  methods  should  not  be  adopted,  but  chemical  titration 
according  to  the  method  already  given  should  be  performed, 
After  titration  the  bouillon  must  again  be  boiled  for  a  few 
minutes,  in  order  to  precipitate  the  acid  albumins,  as  much 
water  added  as  has  been  lost  by  evaporation,  and  the  fluid 
filtered  through  a  pharmaceutic  filter. 

The  filtered  fluid  is  dispensed  in  previously  sterilized  tubes 
with  cotton  plugs — about  10  c.c.  to  each — or  in  flasks,  and 
is  then  sterilized  by  steam  three  successive  days  for  fifteen 

*  "Trans.  Assoc.  Amer.  Phys.,"  1896. 


To  Prepare  Bouillon  from  Meat  Extract       229 

to  twenty  minutes  each,  according  to  the  directions  already 
given  for  intermittent  sterilization,  or  superheated  in  the 
autoclave. 

The  loss  of  water  during  boiling  is  an  important  matter 
to  bear  in  mind,  as  unless  properly  replaced  it  is  the  cause 
of  disproportion  between  the  fluids  and  solids  of  the  media. 
The  quantity  must  therefore  be  measured  before  filtration 
and  enough  water  added  to  replace  what  has  been  lost. 
Measuring  before  filtration  is  comparatively  easy  with 
bouillon,  but  difficult  with  heavy  liquids,  like  the  gelatin 
and  agar-agar  solutions.  To  overcome  this  difficulty  it  is 
best  to  make  the  entire  preparation  by  weight  and  not  by 
volume.  A  pair  of  platform  scales  with  sliding  indicators 
will  first  balance  the  empty  kettle  and  then  show  the  correct 
quantity  of  each  added  ingredient.  After  boiling,  the  kettle 
can  be  returned  to  the  scale  and  the  exact  quantity  of  water 
to  be  added  determined. 

II.  To  Prepare  Bouillon  from  Meat  Extract. — When 
desirable,  the  bouillon  may  also  be  prepared  from  beef- 
extract,  the  method  being  very  simple:  To  1000  c.c.  of 
clean  water  10  grams  of  Witte's  dried  beef-peptone,  5 
grams  of  sodium  chlorid,  and  about  2  grams  of  beef -extract 
are  added.  The  solution  is  boiled  until  the  constituents 
are  dissolved,  titrated,  and  filtered  when  cold.  If  it  be 
filtered  while  hot,  there  is  always  a  subsequent  precipitation 
of  meat-salts,  which  clouds  it. 

Bouillon  and  other  liquid  culture  media  are  best  dis- 
pensed and  kept  in  small  receptacles — test-tubes  or  flasks 
— in  order  that  a  single  contaminating  organism,  should  it 
enter,  may  not  spoil  the  entire  quantity.  A  very  convenient, 
simple  apparatus  used  by  bacteriologists  for  filling  tubes 
with  liquid  media  is  shown  in  figure  45.  It  consists  of  a 
funnel  to  which  a  short  glass  pipet  is  attached  by  a  bit  of 
rubber  tubing.  A  pinch-cock,  at  6,  controls  the  outflow  of 
the  liquid.  The  apparatus  need  not  be  sterilized  before 
using,  as  the  culture  medium  must  subsequently  be  sterilized 
either  by  the  intermittent  method  or  in  the  autoclave  after 
the  tubes  are  filled.  The  test-tubes  and  flasks  into  which 
the  culture  medium  is  filled  must,  however,  be  previously 
sterilized  by  dry  heat,  unless  the  subsequent  sterilization 
is  to  be  performed  in  the  autoclave,  when  it  may  be  un- 
necessary. 


230 


Cultivation  of  Micro-organisms 


Sugar  bouillon  is  bouillon  containing  in  solution  known 
percentages  of  such  sugars  as  glucose,  lactose,  saccharose,  etc. 
As  Smith*  has  pointed  out,  if  the  quantity  of  sugar  in  the 
bouillon  is  to  be  accurately  known,  it  is  necessary  to  first 
destroy  the  muscle  sugars  in  the  meat-infusion  by  adding  a 


Fig.  45. — Funnel  for  filling  tubes  with  culture  media  (Warren) :  a, 
Funnel  containing  the  culture  media  in  liquid  condition ;  b,  pinch-cock 
by  which  the  flow  of  fluid  into  the  test-tube  is  regulated ;  c,  rubbei 
tubing. 

culture  of  the  colon  bacillus  to  the  meat-infusion  and  per- 
mitting fermentation  to  continue  overnight  before  finishing 
the  bouillon  and  adding  the  known  quantity  of  whatever, 
sugar  is  desired.  About  i  per  cent,  of  dextrose,  lactose, 
saccharose  or  galactose  is  all  that  is  required.  More  may  be 
injurious.  If  the  bouillon  be  made  from  meat  extract,  fer- 
mentation may  not  be  necessary. 

The  sugar  bouillons  should  not  be  sterilized  in  the  auto- 
clave, as  the  high  temperatures  chemically  alter  the  sugars. 
*  "Jour,  of  Exp.  Med.,"  n,  No.  5,  p.  546. 


Gelatin  231 


GELATIN. 

The  culture-medium  known  as  gelatin  is  bouillon  to  which 
10  per  cent,  of  gelatin  is  added.  It  has  the  decided  ad- 
vantage over  bouillon  that  it  is  not  only  an  excellent  food 
for  bacteria,  and,  like  the  bouillon,  transparent,  but  also  is 
solid  at  the  room  temperature.  Nor  is  this  all:  it  is  a 
transparent  solid  that  can  be  made  liquid  or  solid  at  will. 
Leffmann  and  La  Wall  have  examined  commercial  gelatins 
and  found  that  many  of  them  contain  sulfur  dioxid  in  quanti- 
ties as  great  as  835  parts  per  million.  As  the  varying  quan- 
tity of  this  impurity  may  modify  the  growth  of  the  culture, 
pure  gelatin  should  be  demanded,  and  all  gelatin  should  be 
washed  for  some  hours  in  cold  running  water  after  being 
weighed  and  before  being  added  to  the  bouillon.  It  is  pre- 
pared as  follows: 

To  1000  c.c.  of  meat-infusion  or  to  1000  c.c.  of  water 
containing  2  grams  of  beef-extract  in  solution,  10  grams 
of  peptone,  5  grams  of  salt,  and  100  grams  of  gelatin  ("Gold 
label"  is  the  best  commercial  article)  are  added,  and  heated 
until  the  ingredients  are  dissolved.  The  solution  reacts 
strongly  acid  and  must  be  corrected  by  titration,  as  already 
described.  It  must  then  be  returned  to  the  fire  and  boiled 
for  about  an  hour.  As  gelatin  is  apt  to  burn  when  boiled 
over  the  direct  flame,  double  boilers  have  been  suggested, 
but  unless  the  outer  kettle  is  filled  with  brine  or  saturated 
calcium  chlorid  solution,  they  are  very  slow,  and  when 
proper  care  is  exercised  there  is  really  no  great  danger  of 
the  gelatin  burning.  It  must  be  stirred  occasionally,  and 
the  flame  should  be  so  distributed  by  wire  gauze  or  by  placing 
a  sheet  of  asbestos  between  it  and  the  kettle  as  not  to  act 
upon  a  single  point.  At  the  end  of  the  hour  the  albumins 
of  the  meat-infusion  will  be  coagulated  and  the  gelatin 
thoroughly  dissolved.  Giinther  has  shown  that  the  gelatin 
congeals  better  if  allowed  to  dissolve  slowly  in  warm  water 
before  boiling.  As  much  water  as  has  been  lost  by  vaporiza- 
tion during  the  process  of  boiling  should  be  replaced.  It 
is  well  to  cool  the  liquid  to  about  60°  C.,  add  the  water  mixed 
with  the  white  of  an  egg  to  clear  the  liquid,  boil  again  for 
half  an  hour,  and  filter. 

If  the  filter  paper  be  of  good  quality,   properly  folded 


232  Cultivation  of  Micro-organisms 

(pharmaceutic  filter),  wet  with  boiling  water,  and  if  the 
gelatin  be  properly  dissolved,  the  whole  quantity  should 
pass  through  before  cooling  too  much.  Should  only  half 
go  through  before  cooling,  the  remainder  must  be  returned 
to  the  pot,  heated  to  boiling  once  more,  and  then  passed 
through  a  new  filter  paper.  As  a  matter  of  fact,  gelatin 
usually  filters  readily.  A  wise  precaution  is  to  catch  the 
first  few  centimeters  in  a  test-tube  and  boil  them,  so  that 
if  cloudiness  show  the  presence  of  uncoagulated  albumin, 
the  whole  mass  can  be  boiled  again.  The  finished  gelatin, 
which  is  perfectly  transparent  and  of  an  amber  color,  is  at 
once  distributed  into  sterilized  tubes  and  sterilized  like  the 
bouillon  by  the  intermittent  method.  The  sterilization  can 
also  be  satisfactorily  performed  by  the  use  of  the  autoclave 
at  i  io°-i  15°  C.  for  fifteen  minutes,  but  this  method  is  prob- 
ably less  well  adapted  to  the  sterilization  of  gelatin  than  of 
the  other  media,  as  the  high  degree  of  heat  injures  its  sub- 
sequent solidifying  power. 

Sterilized  gelatin  or  other  culture  medium  can  be  kept 
en  masse  indefinitely,  but  should  a  contaminating  micro- 
organism accidentally  enter,  the  whole  quantity  will  be 
spoiled ;  if,  on  the  other  hand,  it  be  dispensed  and  kept  in 
tubes,  several  of  them  may  be  contaminated  without  serious 
loss.  When  properly  sterilized  and  protected,  it  should  keep 
indefinitely. 

AGAR-AGAR. 

Agar-agar  is  the  commercial  name  of  a  preparation 
made  from  a  Ceylonese  sea-weed.  It  reaches  the  market 
in  the  form  of  long  shreds  of  semi-transparent,  isinglass- 
like  material,  less  commonly  in  long  bars  of  compressed 
flakes,  rarely  in  the  form  of  powder.  It  dissolves  slowly 
in  boiling  water  with  a  resulting  thick  jelly  when  cold. 
The  jelly,  which  solidifies  between  40°  and  50°  C.,  cannot 
again  be  melted  except  by  the  elevation  of  its  temperature 
to  the  boiling-point.  The  culture  medium  made  from  agar- 
agar  is  nearly  transparent,  and  is  almost  as  useful  as  gelatin, 
as  in  addition  to  its  ability  to  liquefy  and  solidify,  it  has 
the  decided  advantage  of  remaining  solid  at  comparatively 
high  temperatures  so  as  to  permit  keeping  the  cultures  grown 
upon  it  at  the  incubation  temperature, — i.  e.,  37°  C., — at 
which  temperature  gelatin  is  always  liquid. 

The  preparation  of  agar-agar  is  commonly  described  in 


Agar-agar  233 

the  text-books  as  one  "requiring  considerable  patience  and 
much  waste  of  filter  paper."  In  reality,  it  is  not  difficult 
if  a  good  heavy  filter  paper  be  obtained  and  no  attempt 
made  to  filter  the  solution  until  the  agar-agar  is  perfectly 
dissolved. 

It  is  prepared  as  follows:  To  1000  c.c.  of  bouillon  made  as 
described  above,  preferably  of  meat  instead  of  beef -extract, 
10  to  1 5  grams  of  agar-agar  are  added.  The  mixture  is  boiled 
vigorously  for  an  hour  in  an  open  pot  over  the  direct  gas 
flame  or  in  the  double  boiler  with  saturated  calcium  chlorid 
solution  in  the  outside  pot.  After  being  cooled  to  about 
60°  C.,  and  after  the  correction  of  the  reaction  by  titration, 
an  egg  beaten  up  in  water  is  added,  and  the  liquid  again 
boiled  until  the  egg-albumen  is  entirely  coagulated. 

After  the  second  boiling  and  the  replacement  of  the 
volatilized  water,  the  agar-agar  is  filtered  through  a  care- 
fully folded  pharmaceutic  filter  wet  with  boiling  water. 
It  may  expedite  matters  to  pour  in  about  one-half  of  the 
solution,  keep  the  remainder  hot,  and  subsequently  add 
it  when  necessary. 

The  formerly  much  employed  hot-water  and  gas-jet 
filters  are  unnecessary.  If  properly  prepared,  the  whole 
quantity  will  filter  in  from  fifteen  to  thirty  minutes. 

Ravenel  *  prepares  agar-agar  by  making  two  solutions, 
one  representing  the  meat-infusion,  but  twice  the  usual 
strength,  the  other  the  agar-agar  dissolved  in  one-half  the 
usual  quantity  of  water.  The  agar-agar  is  dissolved  by 
exposure  to  superheated  steam  in  the  autoclave,  after  which 
the  two  solutions  are  poured  together  and  boiled  until  all 
of  the  albumins  are  precipitated.  The  coagulation  of  the 
albumins  of  the  meat-infusion  serves  to  clarify  the  agar-agar. 

If  agar-agar  is  to  be  made  with  beef -extract,  the  bouillon 
should  be  made  first  and  filtered  when  cold,  to  exclude  the 
uratic  salts  which  otherwise  precipitate  in  the  agar-agar 
when  cold  and  form  an  unsightly  cloud. 

The  finished  agar-agar  should  be  a  colorless,  nearly  trans- 
parent, firm  jelly.  It  is  dispensed  in  tubes  like  the  gelatin 
and  bouillon,  sterilized  by  steam,  either  by  the  intermittent 
process  or  in  the  autoclave,  and  after  the  last  sterilization, 
before  cooling,  each  tube  is  inclined  against  a  slight  eleva- 
tion, so  as  to  permit  the  jelly  to  solidify  obliquely  and  afford 
an  extensive  flat  surface  for  the  culture. 

*  "  Journal  of  Applied  Microscopy, "June,  1898,  vol.  I,  No.  6,  p.  106. 


234  Cultivation  of  Micro-organisms 

After  the  agar-agar  jelly  solidifies  it  retracts  so  that  a  little 
water  collects  at  the  lower  part  of  the  tube.  This  should  not 
be  removed,  as  it  keeps  the  jelly  moist,  and  also  distinctly 
influences  the  character  of  the  growth  of  the  bacteria. 

Glycerin  Agar=agar. — For  an  unknown  reason  certain 
bacteria  that  will  not  grow  upon  agar-agar  prepared  as  de- 
scribed will  do  so  if  3  to  7  per  cent,  of  glycerin  be  added  after 
filtration.  Among  these  is  the  tubercle  bacillus,  which, }  not 
growing  at  all  upon  plain  agar-agar,  will  grow  well  when 
glycerin  is  added — a  fact  discovered  by  Roux  and  Nocard. 
The  glycerin  added  to  bouillon  or  any  other  medium  has  the 
same  advantageous  influence. 

Blood  Agar=agar  was  recommended  by  R.  Pfeiffer  for 
the  cultivation  of  the  influenza  bacillus.  It  is  ordinary 
agar-agar  whose  surface  is  coated  with  a  little  blood  secured 
under  antiseptic  precautions  from  the  finger-tip,  ear-lobule, 
etc.,  of  man,  or  from  the  vein  of  one  of  the  lower  animals. 
Some  bacteriologists  prepare  a  hemoglobin  agar-agar  by 
spreading  a  little  powdered  hemoglobin  upon  the  surface  of 
the  agar-agar.  This  has  the  disadvantage  that  powdered 
hemoglobin  is  not  sterile,  and  the  medium  must  be  again 
sterilized  after  its  addition. 

The  blood  agar-agar  should  be  kept  in  the  incubator  a  day 
or  two  before  use  so  as  to  insure  perfect  sterility. 

BLOOD-SERUM. 

The  great  advantage  possessed  by  this  medium  is  that  it 
is  itself  a  constituent  of  the  body,  and  hence  offers  oppor- 
tunities for  the  development  of  the  parasitic  forms  of  bac- 
teria. If  the  blood-serum  is  to  be  employed  fresh,  it  must 
either  be  heated  or  kept  sufficiently  long  to  lose  its  natural 
germicidal  properties.  The  statement  that  serum  represents 
the  normal  body-juice  is  erroneous,  as  it  is  minus  the  fibrin 
factors  and  some  of  the  salts,  and  contains  new  bodies  liber- 
ated from  the  destroyed  leukocytes.  Solidified  blood-serum, 
exposed  to  the  heat  of  the  sterilizing  apparatus,  in  no  sense 
resembles  the  body- juices. 

It  is  one  of  the  most  difficult  media  to  prepare.  The 
blood  must  be  obtained  either  by  bleeding  some  good-sized 
animal  or  from  a  slaughter-house  in  appropriate  receptacles, 
the  best  things  for  the  purpose  being  i -quart  fruit  jars  with 
tightly  fitting  lids.  The  jars  are  sterilized  by  heat,  closed, 


Blood-serum  235 

and  carried  to  the  slaughter-house,  where  the  blood  is 
permitted  to  flow  into  them  from  the  severed  vessels  of 
the  animal.  It  seems  advisable  to  allow  the  first  blood 
to  escape,  as  it  is  likely  to  become  contaminated  from 
the  hair.  By  waiting  until  a  coagulum  forms  upon  the  hair 
the  danger  of  contamination  is  obviated.  The  jars,  when 
full,  are  allowed  to  stand  undisturbed  until  firm  coagula 
form  within  them,  after  which  they  are  carried  to  the 
laboratory  and  stood  upon  ice  for  forty:eight  hours,  by 
which  time  the  clots  will  have  retracted  considerably,  and 
a  moderate  amount  of  clear  serum  can  be  removed  by  sterile 
pipets  and  placed  in  sterile  tubes.  If  the  serum  obtained 
be  red  and  clouded  from  the  presence  of  corpuscles,  it  may 
be  pipetted  into  sterile  cylinders  and  allowed  to  sediment 
for  twelve  hours,  then  repipetted  into  tubes.  It  is  evident 
that  such  frequent  manipulations  afford  numerous  chances 
of  infection;  hence  the  sterilization  of ,  the  serum  becomes 
of  the  greatest  importance. 

As  the  demand  for  serum  has  been  considerable  during  the 
last  few  years,  commercial  houses  dealing  in  biologic  pro- 
ducts now  market  fresh  horse  serum,  preserved  with  chloro- 
form, in  liter  bottles.  This  can  be  employed  with  great 
satisfaction,  the  chloroform  being  driven  off  during  coagu- 
lation and  sterilization. 

If  it  be  desirable  to  use  the  serum  as  a  liquid  medium,  it  is 
exposed  to  a  temperature  of  60°  C.  for  one  hour  upon  each 
of  five  consecutive  days.  To  coagulate  the  serum  and  make 
a  solid  culture  medium,  it  may  be  exposed  twice,  for  an 
hour  each  time — or  three  times  if  there  be  reason  to  think  it 
badly  contaminated — to  a  temperature  just  short  of  the 
boiling-point.  During  the  process  of  coagulation  the  tubes 
should  be  inclined,  so  as  to  offer  an  oblique  surface  for  the 
growth  of  the  organisms. .  The  serum  thus  prepared  should 
be  white,  but  may  have  a  reddish-gray  color  if  many  red 
corpuscles  be  present.  It  is  always  opaque  and  cannot  be 
melted ;  once  solid,  it  remains  so. 

Koch  devised  a  special  apparatus  (Fig.  46)  for  coagulating 
blood-serum.  The  bottom  should  be  covered  with  cotton,  a 
single  layer  of  tubes  placed  upon  it,  the  glass  lid  closed  and 
covered  with  a  layer  of  felt,  and  the  temperature  elevated 
until  coagulation  occurs.  The  repeated  sterilizations  may 
be  conducted  in  this  same  apparatus,  or  may  be  done  equally 
well  in  a  steam  apparatus,  the  cover  of  which  is  not  com- 
pletely closed,  for  if  the  temperature  of  the  serum  be  raised 


236  Cultivation  of  Micro-organisms 

too  rapidly  it  is  certain  to  bubble,  so  that  the  desirable 
smooth  surface  upon  which  the  culture  is  to  be  made  is 
ruined. 

Like  other  culture  media,  blood-serum  and  its  combina- 
tions may  be  sterilized  in  the  autoclave  and  much  time 
thus  saved.  The  serum  should,  however,  first  be  coagu- 
lated, else  bubbling  is  apt  to  occur  and  ruin  its  surface. 
The  autoclave  temperature  unfortunately  makes  the  prep- 
aration very  firm  and  hard,  considerable  fluid  being  pressed 
out  of  it. 

It  is  said  that  considerable  advantage  is  secured  from  the 
addition  of  neutrose  to  blood-serum,  which  prevents  its  coag- 


Fig.  46. — Koch's    apparatus   for   coagulating   and   sterilizing    blood- 
serum. 


ulating  when  heated.  It  can  then  be  sterilized  like  bouillon 
and  can  subsequently  be  solidified,  when  desired,  by  the 
addition  of  some  agar-agar. 

Fresh  blood-serum  can  be  kept  on  hand  in  the  laboratory, 
in  sterile  bottles,  by  adding  an  excess  of  chloroform.  In  the 
process  of  coagulation  and  sterilization  the  chloroform  is 
evaporated  ;  the  serum  is  unchanged  by  its  presence. 

Loffler's  Blood-serum  Mixture,  which  seems  rather 
better  for  the  cultivation  of  some  species  than  the  blood- 
serum  itself,  consists  of  i  part  of  a  beef-infusion  bouillon  con- 
taining i  per  cent,  of  glucose  and  3  parts  of  liquid  blood- 
serum.  After  being  well  mixed  the  fluid  is  distributed  in 


Potatoes  237 

tubes,  and  sterilized  and  coagulated  like  the  blood-serum 
itself.  As  prepared  by  Loffler  it  was  soft,  semi-gelatinous 
and  semi-transparent,  not  firm  and  white ;  therefore  should 
be  sterilized  at  low  temperatures.  Many  organisms  grow 
more  luxuriantly  upon  it  than  upon  either  plain  blood-serum 
or  other  culture  media.  Its  especial  usefulness  is  for  the 
cultivation  of  Bacillus  diphtheriae,  which  grows  upon  it 
rapidly  and  with  a  characteristic  appearance. 

Alkaline  Blood=serum. — According  to  Lorrain  Smith,  a 
very  useful  culture  medium  can  be  prepared  as  follows :  To 
each  loo  c.c.  of  blood-serum  add  1-1.5  c-c-  °f  a  IO  per  cent, 
solution  of  sodium  hydrate  and  shake  it  gently.  Put  suffi- 
cient of  the  mixture  into  each  of  a  series  of  test-tubes,  and, 
laying  them  upon  their  sides,  sterilize  like  blood-serum, 
taking  care  that  their  contents  are  not  heated  too  quickly,  as 
then  bubbles  are  apt  to  form.  The  result  should  be  a  clear, 
solid  medium  consisting  chiefly  of  alkali-albumins.  It  is 
especially  useful  for  Bacillus  diphtheriae. 

Deycke's  Alkali=albuminate. — One  thousand  grams  of 
meat  are  macerated  for  twenty-four  hours  with  1200  c.c.  of  a 
3  per  cent,  solution  of  potassium  hydrate.  The  clear  brown 
fluid  is  filtered  off  and  pure  hydrochloric  acid  carefully  added 
while  a  precipitate  forms.  The  precipitated  albuminate  is 
collected  upon  a  cloth  filter,  mixed  with  a  small  quantity  of 
liquid,  and  made  distinctly  alkaline.  To  make  solutions  of 
definite  strength  it  can  be  dried,  pulverized,  and  redissolved. 

The  most  useful  formula  used  by  Deycke  was  a  2.5  per 
cent,  solution  of  the  alkali-albuminate  with  the  addition  of 
i  per  cent,  of  peptone,  i  per  cent,  of  NaCl,  and  gelatin  or 
agar-agar  enough  to  make  it  solid. 

Potatoes. — Without  taking  time  to  review  the  old  method 
of  boiling  potatoes,  opening  them  with  sterile  knives,  and 
protecting  them  in  the  moist  chamber,  or  the  much  more 
easily  conducted  method  of  Esmarch  in  which  the  slices 
of  potato  are  sterilized  in  the  small  dishes  in  which  they 
are  afterward  kept  and  used,  we  will  at  once  pass  to  what 
seems  the  most  simple  and  satisfactory  method — that  of 
Bolton  and  Globig.* 

With  the  aid  of  a  cork -borer  or  Ravenel  potato  cutter 
(Fig.  47)  a  little  smaller  in  diameter  than  the  test-tube  ordi- 
narily used,  a  number  of  cylinders  are  cut  from  potatoes. 
*  "The  Medical  News,"  vol.  i,,  1887,  p.  138. 


238  Cultivation  of  Micro-organisms 

Rather  large  potatoes  should  be  used,  the  cylinders  being 
cut  transversely,  so  that  a  number,  each  about  an  inch  and  a 
half  in  length,  can  be  cut  from  one  potato.  The  skin  is  re- 
moved from  the  cylinders  by  cutting  off  the  ends,  after  which 
each  cylinder  is  cut  in  two  by  an  oblique  incision,  so  as  to 
leave  a  broad,  flat  surface.  The  half-cylinders  are  placed 
each  in  a  test-tube  previously  sterilized,  and  are  exposed 
three  times,  for  half  an  hour  each,  to  the  streaming  steam  of 
the  sterilizer.  This  steaming  cooks  the  potato  and  also 
sterilizes  it.  Such  potato  cylinders  are  apt  to  deteriorate 
rapidly,  first  by  turning  very  dark,  second  by  drying  so  as  to 
be  useless.  Abbott  has  shown  that  if  the  cut  cylinders  be 
allowed  to  stand  for  twelve  hours  in  running  water  before 
being  dispensed  in  the  tubes,  they  are  not  so  apt  to  turn  dark. 
Drying  may  also  be  prevented  by  adding  a  few  drops  of  clean 
water  to  each  tube  before  sterilizing. 
Some  workers  insert  a  bit  of  glass  or  a 
pledget  of  glass  wool  into  the  bottom  of 
the  tube  so  as  to  support  the  potato 
and  keep  it  up  out  of  the  water.  It  is 
not  necessary  to  have  a  special  small 
chamber  blown  in  the  tube  to  contain 
this  water,  only  a  small  quantity  of 
which  need  be  added.  The  special 
reservoir  increases  the  trouble  of  clean- 
Fig.  47,-Ravend's  mg  the  tubes. 

potato  cutter.  If  the  work  to  be  done  with  potatoes 

must  be  accurate,  it  may  be  necessary 
to  correct  their  variable  reaction,  especially  if  the  acids 
have  not  been  sufficiently  removed  by  the  washing  in  run- 
ning water  already  described. 

To  do  this  the  cut  cylinders  are  placed  in  a  measured  quan- 
tity of  distilled  water  and  steamed  for  about  an  hour.  The 
reaction  of  the  water  is  then  determined  by  titration  and 
the  desired  amount  of  sodium  hydroxid  added  to  correct  the 
reaction,  after  which  the  potatoes  are  steamed  in  the  cor- 
rected solution  for  about  thirty  minutes  before  being  placed 
in  the  tubes. 

A  potato-juice  has  also  been  suggested,  and  is  of  some 
value.  It  is  made  thus:  To  300  c.c.  of  water  100  grams  of 
grated  potato  are  added,  and  allowed  to  stand  on  ice  over 
night.  Of  the  pulp,  300  c.c.  are  expressed  through  a  cloth 
and  cooked  for  an  hour  on  a  water-bath.  After  cooking,  the 


Litmus  Milk  239 

liquid  is  filtered,  titrated  if  desired,  and  receives  an  addition 
of  4  per  cent,  of  glycerin.  Upon  this  medium  the  tubercle 
bacillus  grows  well,  especially  when  the  reaction  of  the 
medium  is  acid. 

Milk. — Milk  is  a  useful  culture  medium.  As  the  cream 
which  rises  to  the  top  is  a  source  of  inconvenience,  it  is  best 
to  secure  fresh  milk  from  which  the  cream  has  been  removed 
by  a  centrifugal  machine.  It  is  given  the  desired  degree  of 
alkalinity  by  titration,  dispensed  in  sterile  tubes,  and  ster- 
ilized by  steam  by  the  intermittent  method  or  in  the  auto- 
clave. The  opaque  nature  of  this  culture  medium  often 
permits  the  undetected  development  of  contaminating  or- 
ganisms. A  careful  watch  should  therefore  be  kept  lest  it 
spoil. 

Litmus  Milk. — This  is  milk  to  which  just  enough  of  a 
saturated  watery  solution  of  pulverized  litmus  is  added  to 
give  a  distinct  blue  color  after  titration.  Litmus  milk  is 
probably  the  best  reagent  for  determining  acid  and  alkali 
production  by  bacteria. 

The  watery  solution  of  litmus,  being  a  vegetable  infusion, 
is  likely  to  be  spoiled  by  micro-organismal  growth,  hence 
must  be  treated  like  the  culture  media  and  sterilized  by 
steam  every  time  the  receptacle  in  which  it  is  kept  is 
opened. 

An  excellent  method  of  preparing  litmus  is  given  by 
Prescott  and  Winslow*  and  is  as  follows: 

To  one-half  pound  of  litmus  cubes  add  enough  water  to 
more  than  cover,  boil,  decant  off  the  solution.  Repeat  this 
operation  with  successive  small  quantities  of  water  until 
3  to  4  liters  of  water  have  been  used  and  the  cubes  are  well 
exhausted  of  coloring  matter.  Pour  the  decantations 
together  and  allow  them  to  settle  over  night.  Siphon  off 
the  clear  solution.  Concentrate  to  about  i  liter  and  make 
the  solution  decidedly  acid  with  glacial  acetic  acid.  Boil 
down  to  about  ^  liter  and  make  exactly  neutral  with  caustic 
soda  or  potash.  To  test  for  the  neutral  point,  place  one 
drop  of  the  solution  in  a  test-tube,  while  one  drop  of  ^r  HC1 
should  turn  it  red,  one  drop  of  ^  NaOHO  should  turn  it 
blue.  Filter  the  solution  and  sterilize  at  no°C.  This 
solution  should  be  added  to  the  media  just  before  use  in 
the  proportion  of  about  \  c.c.  to  5  c.c.  of  medium. 

*  "Elements  of  Water  Bacteriology,"  John  Wiley  &  Sons,  New  York, 
1904,  p.  126. 


240  Cultivation  of  Micro-organisms 

If  litmus  be  added  to  the  milk  before  sterilization,  it  is  apt 
to  be  browned  or  decolorized,  so  that  it  is  better  to  sterilize 
the  two  separately  and  pour  them  together  subsequently. 
It  is  said  that  lacmoid  is  never  thus  changed,  and  many 
workers  prefer  it  to  litmus  on  that  account. 

Petruschky's  Whey. — In  order  to  differentiate  between 
acid  and  alkali  producers  among  the  bacteria,  Petruschky 
has  recommended  a  neutral  whey  colored  with  litmus.  It  is 
made  as  follows: 

To  a  liter  of  fresh  skimmed  milk  i  liter  of  water  is  added. 
The  mixture  is  violently  shaken.  About  10  c.c.  are  taken 
out  as  a  sample  to  determine  how  much  hydrochloric  acid 
must  be  added  to  produce  coagulation  of  the  milk,  and, 
having  determined  the  least  quantity  required  for  the  whole 
bulk,  it  is  added.  After  coagulation  the  whey  is  filtered  off, 
exactly  neutralized,  and  boiled.  After  boiling  it  is  found 
clouded  and  acid  in  reaction.  It  is  therefore  filtered  again, 
and  again  neutralized.  Litmus  is  finally  added  to  the  neu- 
tral liquid,  so  that  it  has  a  violet  color,  changed  to  blue  or 
red  by  alkalies  or  acids. 

The  medium  is  a  very  useful  aid  in  differentiating  the 
typhoid  and  colon  bacilli,  showing  well  the  alkali  formation 
of  the  former  and  acid  of  the  latter. 

Peptone  Solution,  or  Dunham's  solution,  is  useful  for  the 
detection  of  certain  faint  colors.  It  is  a  perfectly  clear, 
colorless  solution,  made  as  follows: 

Sodium  chlorid 0.5 

Witte's  dried  peptone 1.0 

Water 100.0 

Boil  until  the  ingredients  dissolve ;  filter,  fill  into  tubes  and  sterilize. 

It  was  for  a  long  time  used  for  the  detection  of  indol. 
Garini  *  found  that  many  of  the  peptones  upon  the  market 
were  impure,  and  on  this  account  failed  to  show  the  indol 
reaction  in  cultures  of  bacteria  known  to  produce  it.  He 
recommends  testing  the  peptone  to  be  employed  by  the  use 
of  the  biuret  reaction.  The  reagent  employed  is  Fehling's 
copper  solution,  with  which  pure  peptone  strikes  a  violet 
color  not  destroyed  upon  boiling,  while  impure  peptone 
gives  a  red  or  reddish- yellow  precipitate.  Both  the  peptone 
and  copper  solutions  should  be  in  a  dilute  form  to  make 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  xm,  p.  790. 


Peptone  Solution  241 

successful  tests.  The  addition  of  4  c.c.  of  the  following 
solution — 

Rosolic  acid    0.5 

Eighty  per  cent,  alcohol  .  .  .  , 100.0 

makes  the  peptone  solution  a  reagent  for  the  detection  of 
acids  and  alkalies.  The  solution  is  of  a  pale  rose  color. 
If  the  organisms  cultivated  produce  acids,  the  color  fades ; 
if  alkalies,  it  intensifies.  As  the  color  of  rosolic  acid  is  de- 
stroyed by  glucose,  it  cannot  be  used  in  culture  media  con- 
taining it. 

Theobald  Smith*  has  called  attention  to  the  fact  that 
many  bacteria  fail  to  grow  in  Dunham's  solution,  and 
recommends  that,  for  the  detection  of  indol,  bouillon  free  of 
dextrose  be  used  instead.  All  bacteria  grow  well  in  it,  and 
the  indol  reaction  is  pronounced  in  sixteen-hour-old  cultures. 
His  method  of  preparation  is  as  follows:  Beef -infusion,  pre- 
pared either  by  extracting  in  the  cold  or  at  60°  C.,  is  inocu- 
lated in  the  evening  with  a  rich  fluid  culture  of  some  acid- 
producing  bacterium  (Bacillus  coli)  and  placed  in  the  ther- 
mostat. Early  next  morning  the  infusion,  covered  with  a 
thin  layer  of  froth,  is  boiled,  filtered,  peptone  and  salt  added, 
and  the  neutralization  and  sterilization  carried  on  as  usual. 

This  method  is  subject  to  error  caused  by  the  presence  in 
the  medium  of  indol  produced  by  the  colon  bacillus.  This 
can  be  demonstrated  if  the  tests  for  indol  be  sensitive. 
Selterf  finds  that  the  method  of  Smith  gives  inferior  results 
to  a  simple  culture-medium  consisting  of  water,  90  parts; 
Witte's  peptone,  10  parts;  sodium  phosphate,  0.5  part,  and 
magnesium  sulphate,  o.i  part. 

Other  culture-media  employed  for  special  purposes  will  be 
mentioned  as  occasion  arises. 

*  "Journal  of  Kxp.  Medicine,"  Sept.  5,  1897,  vi,  p.  546. 
f'Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Orig.  u,  p.  465- 

16 


CHAPTER  VIII. 
CULTURES,  AND  THEIR  STUDY. 

THE  purposes  for  which  culture  media  are  prepared  are 
numerous.  Through  their  aid  it  is  possible  to  isolate  the 
micro-organisms,  to  keep  them  in  healthy  growth  for  con- 
siderable lengths  of  time,  during  which  their  biologic  peculiar- 
ities can  be  observed  and  their  metabolic  products  collected, 
and  to  introduce  them  free  from  contamination  into  the 
bodies  of  experiment  animals. 

The  isolation  of  bacteria  was  next  to  impossible  until  the 
fluid  media  of  the  early  observers  were  replaced  by  the  solid 
culture  media  introduced  by  Koch,  and  exceedingly  diffi- 
cult until  he  devised  the  well-known  "plate  cultures." 

A  growth  of  artificially  planted  micro-organisms  is  called 
a  culture.  If  such  a  growth  contains  but  one  kind  of  organ- 
ism, it  is  known  as  a  pure  culture. 

It  has  at  present  become  the  custom  to  use  the  term  "cul- 
ture" rather  loosely,  so  that  it  does  not  always  signify  an 
artificially  planted  growth  of  micro-organisms,  but  may 
signify  a  growth  taking  place  under  natural  conditions ;  thus, 
typhoid  bacilli  are  said  to  occur  in  "  pure  culture"  in  the 
spleens  of  patients  dead  of  that  disease,  because  no  other 
bacteria  are  associated  with  them ;  and  sometimes,  when  the 
tubercle  bacilli  are  very  numerous  and  unmixed  with  other 
bacteria,  in  the  expectorated  fragments  of  cheesy  matter 
from  tuberculosis  pulmonalis,  they  are  said  to  occur  in 
"pure  culture." 

The  culture  manipulations  are  performed  either  with  a 
sterilized  platinum  wire  or  with  a  capillary  pipet  of  glass. 

The  platinum  wire  is  so  limber  that  it  is  scarcely  to  be 
recommended,  and  a  wire  composed  of  platinum  and  iridium, 
which  is  elastic  in  quality,  is  to  be  preferred.  The  wires 
are  about  5  cm.  in  length,  of  various  thicknesses  according  to 
the  use  for  which  they  are  employed,  and  are  usually  fused 
into  a  thin  glass  rod  about  17  cm.  in  length  (Fig.  48).  The 
wires  may  be  straight  or  provided  with  a  small  loop  at  the 
end  so  as  to  conveniently  take  up  small  drops  of  fluid. 

242 


Technic  of  Culture  Manipulation  243 

Heavy  wires  used  for  securing  diseased  tissue  from  animals 
may  be  flattened  at  the  ends  by  hammering,  and  may  thus 
be  fashioned  into  miniature  knives,  scrapers,  harpoons,  etc., 
as  desired. 

Ravenel  has  invented  a  convenient  form  for  carrying  in 
the  pocket.  It  consists  of  the  platinum  wire  fastened  in  a 
heavier  aluminium  wire  which  in  turn  fits  into  a  piece  of 
glass  tubing.  When  carried  in  the  pocket,  the  position  of 


Fig.    48. — Platinum    needles   for   transferring   bacteria;    made   from 
No.  27  platinum  wire  inserted  in  glass  rods. 

the  platinum  wire  is  reversed  in  the  glass  tubing  and  pro- 
tected by  it  (Fig.  49). 

Immediately  before  and  immediately  after  use,  the  platinum 
wire  is  to  be  sterilized  by  heating  to  incandescence  in  a  flame, 
in  order  that  it  convey  nothing  undesirable  into  the  culture, 
and  in  order  that  it  scatter  no  micro-organisms  about  the 
laboratory. 

Capillary  glass  tubes  are  employed  by  the  French  for 
many  of  the  manipulations.  They  are  made  of  J-  or  f-inch 
glass  tubing  cut  into  25  cm.  lengths,  heated  at  the  center, 


Fig.  49. — Platinum  wires  for  bacteriologic  use. 

and  drawn  out  to  capillary  ends  about  5  cm.  long.  They 
are  sealed  at  one  end  and  plugged  with  cotton  at  the  other, 
and  a  number  of  them,  prepared  at  the  same  time,  sterilized 
(Fig.  50).  They  can  be  used  for  all  the  purposes  for  which 
the  platinum  wire  is  employed,  and  in  addition  can  be  used 
as  containers  for  small  quantities  of  fluids  sealed  in  them. 
When  about  to  use  such  a  tube,  its  sealed  capillary  end 
should  be  broken  off  with  forceps,  and  the  tube  sterilized 
by  flaming. 

Technic  of  Culture  Manipulation. — In  order  that  ac- 
curate results  may  accrue  from  the  employment  of  culture 


244  Cultures,  and  their  Study 

media,  and  that  cultures  planted  in  them  may  not  be  con- 
taminated through  improper  technic,  it  is  important  habit- 
ually to  practise  certain  manipulations  by  which  as  much 
latitude  can  be  given  the  operator  as  is  consistent  with 
thorough  defense  against  contaminating  organisms.  To  this 
end  the  containers  of  stored  culture  media  should  be  kept  in 
an  upright  position,  that  the  cotton  stoppers  are  not  mois- 
tened or  soiled.  If  moistened  with  the  culture  media,  molds 
whose  spores  fall  upon  the  surface  of  the  stoppers  may 
gradually  work  their  mycelial  threads  between  the  fibers 
until  they  appear  upon  the  inner  surface  and  drop  newly 
formed  spores  into  the  contained  media,  or  the  cotton  stop- 
pers may  be  glued  fast  and  further  successful  manipulations 
prevented. 

In  handling  tubes  care  must  be  taken  to  stand  them  up  in 
tumblers,  racks,  or  other  contrivances,  and  not  lay  them 
upon  the  table  so  that  the  contents  touch  the  stoppers. 


o 


Fig.  50. — Capillary  glass  tubes. 

When  the  cotton  plugs  are  removed  in  order  that  the 
contents  of  the  tubes  or  flasks  may  be  inoculated  or  other- 
wise manipulated  the  removal  and  replacement  should  be 
done  as  quickly  as  convenient,  and  the  mouth  of  the  tube 
should  be  flamed  before  removal.  They  must  be  held 
between  the  fingers,  by  that  part  which  projects  above  the 
glass,  not  laid  upon  the  table,  from  which  dust,  and  inci- 
dentally bacteria,  may  be  taken  up  and  subsequently 
dropped  into  the  medium;  nor  must  they  be  touched  with 
the  fingers  at  that  part  which  enters  the  neck  of  the  container 
lest  they  take  up  micro-organisms  from  the  skin.  The 
stoppers  thus  require  careful  consideration  lest  they  become 
the  source  of  future  contamination. 

So  soon  as  the  cotton  stopper  is  removed,  the  medium  is 
left  without  protection  from  whatever  micro-organisms 
happen  to  be  in  the  air,  so  that  it  should  be  replaced  as 
soon  as  possible,  and  every  manipulation  requiring  its 
removal  performed  expeditiously.  During  the  time  the 


Technic  of  Culture  Manipulation  245 

stopper  is  withdrawn  it  is  wise  to  hold  the  tubes  or  other 
containers  in  an  oblique  or  horizontal  position  that  will  aid 
in  excluding  the  micro-organisms  of  the  air.  Thus,  a  tube 
held  vertically  can  probably  more  easily  receive  such  organ- 
isms than  one  held  horizontally  or  reversed.  Some  bacteri- 
ologists make  inoculations  with  the  tubes  reversed  in  all  cases 
in  which  solid  media  are  employed,  but  it  is  not  at  all  neces- 
sary. If  the  tubes  are  held  obliquely,  the  danger  of  con- 
tamination is  reduced  to  a  minimum.  It  is  well  to  adopt 
some  method  of  handling  the  tubes  that  has  given  satis- 
faction to  others  and  is  found  convenient  to  one's  self  and 
habitually  practise  it  until  it  becomes  second  nature  and 
can  be  done  without  thought. 

The  usual  method  of  making  a  transplantation  of  bacteria 
from  culture-tube  to  cul- 
ture-tube is,  in  detail,  as 
follows : 

In  order  that  any  bac- 
teria loosely  scattered 
over  the  surface  of  the 
cotton  stopper,  and  upon 
the  glass  near  the  mouth 
of  the  tube,  may  be  de- 
stroyed and  prevented 
from  entering  the  med- 
ium as  the  stopper  is  with- 
drawn, both  the  tube  con- 
taining the  culture  and 
the  fresh  tube  to  which  it 
is  to  be  transferred  should 
be  held  for  a  moment  in 
a  flame  and  rolled  from  side  to  side  so  that  all  parts  are  flamed. 
The  cotton  ignites  and  blazes  actively,  but  the  flame  can 
be  extinguished  by  forcibly  blowing  upon  it  and  any  smolder- 
ing remains  extinguished  by  pinching  with  the  fingers.  The 
tubes  are  now  placed  side  by  side  between  the  thumb  and 
upward-directed  palm  of  the  left  hand,  the  stoppers  toward 
the  operator.  The  position  of  the  tubes  should  be  such  as 
to  permit  one  to  see  the  contained  media  without  the  fingers 
being  in  the  way.  The  stopper  of  the  tube  toward  the 
left  is  removed  by  a  gentle  twist  and  placed  between  the 
index  and  middle  fingers  of  the  left  hand;  the  stopper  of 
the  next  tube  similarly  removed  and  placed  between  the 


Fig.  51. — Method  of  holding  tubes 
during  inoculation. 


246  Cultures,  and  their  Study 

middle  and  ring  fingers  of  the  same  hand  (Fig.  51).  If  three 
or  four  tubes  are  to  be  held,  the  third  stopper  can  be  placed 
between  the  ring  and  little  fingers  of  the  left  hand  and  the 
fourth  retained  in  the  right  hand.  The  part  of  each  stopper 
that  enters  the  tube  must  not  be  touched. 

The  necessary  manipulation  is  usually  made  with  the 
platinum  wire,  which  is  sterilized  by  heating  to  incandes- 
cence before  using.  The  wire  must  not  be  used  while  hot, 
but  cools  in  a  moment  or  two.  The  culture  is  touched,  the 
wire  entering  and  exiting  without  touching  the  tube,  and 
the  bacteria  adhering  to  the  wire  are  applied  to  the  medium 
in  the  other  tube,  the  same  care  being  exerted  not  to  have 
the  platinum  wire  touch  the  glass.  After  the  transfer  is 
made,  the  wire  is  made  incandescent  in  the  flame  before 
being  returned  to  the  table  or  stand  made  to  hold  it,  and 
the  stoppers  returned  one  after  the  other,  each  to  its  own 
tube,  that  part  entering  the  tube  riot  being  touched.  Each 
stopper  is  given  a  twist  as  it  -enters  the  mouth  of  the  tube. 

Modifications  of  tfiese  directions  can  be  made  to  suit  the 
different  forms  of  containers  used,  but  the  essential  features 
must  be  maintained. 

When  any  manipulation  requires  that  a  tube  or  flask  be 
permitted  to  remain  open  an  unusual  length  of  time,  its 
contamination  from  the  air  can  be  prevented  for  some 
minutes  by  heating  its  neck  quite  hot.  The  air  about  it, 
being  heated  by  the  hot  glass,  ascends,  forming  a  current 
that  carries  the  bacteria  away  from,  rather  than  into,  the 
receptacle. 

Isolation  of  Bacteria. — Three  principal  methods  are,  at 
present,  employed  for  securing  pure  cultures  of  bacteria. 
Before  beginning  a  description  of  them  it  is  well  to  observe 
that  the  peculiarities  of  certain  pathogenic  micro-organisms 
enable  us  to  use  special  means  for  their  isolation,  and  that 
these  general  methods  are  chiefly  useful  for  the  isolation  of 
non-pathogenic  organisms. 

Plate  Cultures. — All  the  methods  depend  upon  the  obser- 
vation of  Koch,  that  when  bacteria  are  equally  distributed 
throughout  some  liquefied  nutrient  medium  that  is  subse- 
quently solidified  in  a  thin  layer,  they  grow  in  scattered 
groups  or  families,  called  colonies,  distinctly  isolated  from 
one  another  and  susceptible  of  transplantation. 

The  plate  cultures,  as  originally  made  by  Koch,  require 
considerable  apparatus,  and  of  late  years  have  given  place 
to  the  more  ready  methods  of  Petri  and  von  Ksmarch. 


Isolation  of  Bacteria  247 

So  great  is  their  historic  interest,  however,  that  it  would  be  a 
great  omission  not  to  describe  the  original  method  in  detail. 
Apparatus.— Half  a  dozen  glass  plates,  measuring  about 
6  by  4  inches/free  from  bubbles  and  scratches  and  ground  at 
the  edges,  are  carefully  cleaned,  placed  in  a  sheet-iron  box 
made  to  receive  them,  and  sterilized  in  the  hot-air  closet. 
The  box  is  kept  tightly  closed,  and  in  it  the  sterilized  plates 
can  be  kept  indefinitely  before  use. 

A  moist  chamber,  or  double  dish,  about  10  inches  in  diam- 
eter and  3  inches  deep,  the  upper  half  being  just  enough 
larger  than  the  lower  to  allow  it  to  close  over  it,  is  carefully 
washed.  A  sheet  of  bibulous  paper  is  placed  in  the  bottom, 
so  that  some  moisture  can  be  retained,  and  a  i :  1000  bichlorid 
of  mercury  solution  poured  in  and  brought  in  contact  with 
the  sides,  top,  and  bottom 
by  turning  the  dish  in  all 
directions.  The  solution  is 
emptied  out,  and  the  dish, 
which  is  kept  closed,  is 
ready  for  use. 

A  leveling  apparatus  is 
required  (Fig.  52).  It  con- 
sists of  a  wooden  tripod  with 
adjustable  screws,  and  a 
glass  dish  covered  by  a  flat 
plate  of  glass  upon  which  a  Fig-  52. — Complete  leveling  ap- 
low  bell- jar  stands.  The  ESS£b?K™8  Pla*  <"*•»». 
glass  dish  is  filled  with 

broken  ice  and  water,  covered  with  the  glass  plate,  and  then 
exactly  leveled  by  adjusting  the  screws  under  the  legs  of 
the  tripod.  When  level,  the  cover  is  placed  upon  it,  and  it 
is  ready  for  use. 

Method. — A  sterile  platinum  loop  is  dipped  into  the  mate- 
rial to  be  examined,  a  small  quantity  secured,  and  stirred 
about  so  as  to  distribute  it  evenly  throughout  the  con- 
tents of  a  tube  of  melted  gelatin.  If  the  material  under 
examination  be  very  rich  in  bacteria,  one  loopful  may  con- 
tain a  million  individuals,  which,  if  spread  out  in  a  thin 
layer,  would  develop  so  many  colonies  that  it  would  be 
impossible  to  see  any  one  clearly;  hence  further  dilution 
becomes  necessary.  From  the  first  tube,  therefore,  a  loop- 
ful of  gelatin  is  carried  to  a  second  and  stirred  well,  so  as 
to  distribute  the  organisms  evenly  throughout  its  contents. 


248  Cultures,  and  their  Study 

In  this  tube  we  may  have  no  more  than  ten  thousand  organ- 
isms, and  if  the  same  method  of  dilution  be  used  again,  the 
third  tube  may  have  only  a  few  hundreds,  and  a  fourth  only 
a  few  dozen  colonies. 

After  the  tubes  are  thus  inoculated,  one  of  the  sterile  glass 
plates  is  caught  by  its  edges,  removed  from  the  iron  box,  and 
placed  beneath  the  bell-glass  upon  the  cold  plate  covering 
the  ice- water  of  the  leveling  apparatus.  The  plug  of  cotton 
closing  the  mouth  of  tube  No.  i  is  removed,  and  to  prevent 
contamination  during  the  outflow  of  the  gelatin  the  mouth  of 
the  tube  is  held  in  the  flame  of  a  Bunsen  burner  for  a  moment 
or  two.  The  gelatin  is  then  cautiously  poured  out  upon  the 
plate,  the  mouth  of  the  tube,  as  well  as  the  plate,  being 
covered  by  the  bell-glass  to  prevent  contamination  by  germs 
in  the  air.  The  apparatus  being  level,  the  gelatin  spreads 
out  in  an  even,  thin  layer,  and,  the  plate  being  cooled  by  the 
ice  beneath,  it  immediately  solidifies,  and  in  a  few  moments 
can  be  removed  to  the  moist  chamber  prepared  to  receive  it* 

As  soon  as  plate  No.  i.  is  pre- 
pared, the  contents  of  tube  No. 
2  are  poured  upon  plate  No. 
2,  allowed  to  spread  out  and 

Fig.  53.-Glass  bend^  solidify,  and  then  superimposed 

on  plate  No.  i  in  the  moist 

chamber,  being  separated  from  the  plate  already  in  the 
chamber  by  small  glass  benches  (Fig.  53)  made  for  the  purpose 
and  previously  sterilized.  After  the  contents  of  all  the  tubes 
are  thus  distributed,  the  moist  chamber  and  its  contents  are 
stood  away  to  permit  the  bacteria  to  grow.  Where  each 
organism  falls  a  colony  develops,  and  the  success  of  the 
whole  method  depends  upon  the  isolation  of  a  colony  and  its 
transfer  to  a  tube  of  new  sterile  culture  media,  where  it  can 
grow  unmixed  and  undisturbed. 

From  the  description  it  must  be  evident  that  only  those 
culture  media  that  can  be  melted  and  solidified  at  will  can  be 
used  for  plate  cultures — viz.,  gelatin,  agar-agar,  and  glycerin 
agar-agar.  Blood-serum  and  Loffler's  mixture  are  entirely 
inappropriate. 

The  chief  drawbacks  to  this  excellent  method  are  the 
cumbersome  apparatus  required  and  the  comparative  im- 
possibility of  making  plate  cultures,  as  is  often  desirable,  in 
the  clinic,  at  the  bedside,  or  elsewhere  than  in  the  labora- 
tory. The  method  therefore  soon  underwent  modifications,. 


Petri's  Dishes 


249 


the  most  important  being  that  of  Petri,  who  invented  special 
dishes  to  be  used  instead  of  plates. 

Petri's  Dishes.— These  are  glass  dishes,  about  4  inches 
in  diameter  and  J  inch  deep,  with  accurately  fitting  lids. 
They  were  first  recommended  by  Petri*  and  greatly  sim- 
plify bacteriologic  technic  by  dispensing  with  the  plates  and 
plate-boxes,  the  moist  chambers  and  benches,  and  usually 


Fig.  54. — Petri  dish  for  making  plate  cultures. 

with  the  levelling  apparatus  of  Koch,  though  this  is  still  em- 
ployed in  some  laboratories,  and  must  always  be  employed 
when  an  even  distribution  of  the  colonies  is  necessary  in 
order  that  they  can  be  accurately  counted. 

The  method  of  using  the  Petri  dishes  is  very  simple.  They 
are  carefully  cleaned,  polished,  and  sterilized  by  hot  air,  care 
being  taken  that  they  are  placed  in  the  hot-air  closet  right 
side  up,  and  after  sterilization  are  kept  covered  and  in  that 


Fig-  55- — Petri  dish  forceps. 

position.  They  should  be  sterilized  immediately  before 
using,  or  if  they  must  be  kept  for  a  time  should  be  wrapped 
in  tissue  paper  and  then  sterilized.  The  tissue  paper  pro- 
tects the  interior  from  the  accidental  entrance  of  dust  be- 
tween dish  and  lid,  keeps  the  dish  closed,  and  need  not  be 
removed  until  the  last  moment  before  using. 

Time  can  be  saved  by  sterilizing  the  dish  and  cover  in 
the  direct  flame,  instead  of  in  the  hot-air  closet,  special 
forceps  (Fig.  55)  adapted  to  holding  them  having  been 
devised  by  Rosenberger.* 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  i,  No.  i,  1887,  p.  279. 

t  "Phila.  Med.  Jour.,"  Oct.  20,  1900,  vol.  vi,  No.  16,  p.  760. 


25° 


Cultures,  and  their  Study 


The  dilution  of  the  material  under  examination  is  made 
with  gelatin  or  agar-agar  tubes  in  the  manner  above  de- 
scribed, the  plug  is  removed,  the  mouth  of  the  tube  cau- 
tiously held  for  a  moment  in  the  flame,  and  the  contents 
poured  into  one  of  the  sterile  dishes,  whose  lid  is  just  suffi- 
ciently elevated  to  permit  the  mouth  of  the  tube  to  enter. 
The  gelatin  is  spread  over  the  bottom  of  the  dish  in  an 
even  layer,  allowed  to  solidify,  labeled,  inverted,  so  that  the 
water  of  condensation  may  not  drop  from  the  lid  upon  the 
culture  film  and  spoil  the  cultures,  and  stood  away  for  the 
colonies  to  develop. 

To  overcome  the  difficulty  of  excessive  water  of  conden- 
sation Hill  has  introduced  lids  made  of  porous  clay,  by  which 
the  moisture  is  absorbed.  These  can  be  obtained  from 
most  laboratory  purveyors. 

Among  the  other  advantages  of  the  Petri  dish  is  the 
convenience  with  which  colonies  can  be  studied  with  a  low- 
power  lens.  To  do  this  with  the  Koch  plates  meant  to 
remove  them  from  the  sterile  chamber  to  the  stage  of  a 
microscope  and  so  expose  them  to  the  air,  and  to  con- 
tamination, but  to  examine  colonies  in  the  Petri  dish,  one 
simply  examines  through  the  thin  glass  of  the  bottom  dish 
without  any  exposure  to  contaminating  organisms. 

Es march's  Tubes. — This  method,  devised  by  Esmarch, 
converts  the  wall  of  the  test-tube  into  the  plate  and  dispenses 


Fig.  56. — Esmarch  tube  on  block  of  ice  (redrawn  after  Abbott). 

with  all  other  apparatus.  The  tubes,  which  are  inoculated 
and  in  which  the  dilutions  are  made,  should  contain  less  than 
half  the  usual  amount  of  gelatin  or  agar-agar.  After  inocu- 


Colonies 


251 


lation  the  cotton  plugs  are  pushed  into  the  tubes  until  even 
with  their  mouths,  and  then  covered  with  a  rubber  cap,  which 
protects  them  from  wetting.  A  groove  is  next  cut  in  a  block 
of  ice,  and  the  tube,  held  almost  horizontally,  is  rolled  in  this 
until  the  entire  surface  of  the  glass  is  covered  with  a  thin 
layer  of  the  solidified  medium  (Fig.  56).  Thus  the  tube  itself 
becomes  the  plate  upon  which  the  colonies  develop. 

In  carrying  out  Esmarch's  method,  the  tube  must  not  con- 
tain too  much  of  the  culture  medium,  or  it  cannot  be  rolled 
into  an  even  layer;  the  contents  should  not  touch  the  cotton 
plug,  lest  it  be  glued  to  the  glass  and  its  subsequent  useful- 
ness injured,  and  no  water  must  be  admitted  from  the  melted 
ice. 

Colonies. — The  progeny  of  each  bacterium  form  a  mass 


57- — Types  of  colonies:  a,  Cochleate  (B.  coli,  abnormal  form); 
6,  conglomerate  (B.  zopfii) ;  c,  ameboid  (B.  vulgatus) ;  d,  filamentous 
(Frost). 


Fig.  58. — Surface  elevations  of  growths:  a,  Flat;  b,  raised;  c,  convex; 
d,  pulvinate;  e,  capitate;  /,  umbilicate;  g,  umbonate  (Frost), 

which  is  known  as  a  colony.  When  these  are  separated  from 
one  another,  each  is  spoken  of  as  a  single  colony,  and  dif- 
ferent characteristics  belonging  to  different  micro-organisms 
enable  us  at  times  to  recognize  by  macroscopic  and  micro- 
scopic study  of  the  colony  the  particular  kind  of  micro- 
organism from  which  it  has  grown.  The  foregoing  illustra- 
tions show  the  various  types  of  colonies  and  the  legends 
the  terms  used  in  describing  them  (Fig.  57). 

Growing  colonies  should  be  observed  from  day  to  day,  as 
it  not  infrequently  happens  that  unexpected  changes,  such 
as  pigmentation  and  liquefaction,  develop  after  the  colony 


252 


Cultures,  and  their  Study 


is  several  days  old,  and  indeed  sometimes  not  until  much 
later.  Again,  many  colonies  make  their  first  appearance  as 
minute,  sharply  circumscribed  points,  and  later  spread  upon 
the  surface  of  the  culture  medium,  either  in  the  form  of  a 
thin,  homogeneous  layer  or  a  filamentous  cluster.  It  is 
particularly  important  that  in  describing  new  species  of 
bacteria  an  account  of  the  appearance  of  the  colonies  from 
day  to  day,  comparing  all  of  their  variations  for  at  least 
two  weeks,  should  be  included. 

Pure  Cultures. — Single  colonies  also  subserve  a  second 
very  important  purpose,  that  of  enabling  us  to  secure  pure 
cultures  of  bacteria  from  a  mixture.  The  usual  method  of 
doing  this  is  by  the  use  of  plates,  Petri  dishes,  or  Esmarch 
tubes  according  to  the  methods  already  described.  For 


Fig-  59- — Microscopic  structure  of  colonies:  1,  Areolate ;  2,  grumosej 
3,  moruloid;  4,  clouded;  5,  gyrose;  6,  marmorated ;  7,  reticulate, 
8,  repand;  9,  lobate;  10,  erose;  11,  auriculate;  12,  lacerate;  13,  fim- 
bricate;  14,  ciliate  (Frost). 


this  purpose  an  isolated  colony  is  selected  and  carefully 
examined  to  see  that  it  is  single  and  not  a  mixture  of  two 
closely  approximated  colonies  of  different  kinds,  and  then 
transplanted  to  a  tube  of  an  appropriate  culture  medium. 
If  the  colonies  are  few  and  of  good  size,  each  is  transplanted 
to  the  medium  directly  and  without  instrumental  assistance. 
If,  however,  the  colonies  are  numerous,  of  small  size,  and 
close  together,  it  may  be  necessary  to  do  it  under  a  dis- 
secting microscope  or  even  a  low  power  of  the  ordinary 
bacteriologic  microscope.  This  operation  of  transplantation 
is  familiarly  known  as  fishing. 

Fishing. — It  requires  considerable  practice  and  skill  to 
fish  successfully,  and  the  student  should  early  begin  to 
practise  it.  The  colony  to  be  transplanted,  selected  because 
of  its  isolation,  its  typical  appearance,  and  convenient  posi- 


Fishing  for  Colonies  253 

tion  on  the 'plate,  is  brought  to  the  center  of  the  field  and  the 
plate  firmly  held  in  position  with  the  left  hand.     A  sterile 
platinum  wire  is  held  in  the  right  hand,  the  little  finger,  com- 
fortably fixed  upon  the  stage  of  the  microscope,  being  used  to 
support  the  hand.     As  the  operator  looks  into  the  micro- 
scope the  point  of  the  platinum  wire  is  carefully  brought 
into  the  field  of  vision  without  touching  either  the  lens  of 
the  microscope  or  any  part  of  the  plate  beneath.     Of  course, 
the  wire  and  the  colony  cannot  be  simultaneously  looked 
upon.     When   the  colony  is   distinctly  seen  the  platinum 
wire  appears  as  a  shadow,  but  the  endeavor  should  be  to 
make  the  end  of  the  shadow  which  corresponds  to  the  point 
of  the  wire  appear  exactly  over  the  colony.     It  is   then 
gradually  depressed  until  it  touches  the  colony  and  can  be 
seen  to  break  up  and  remove  some  of  its  substance;    or 
should  the  colony  be  tough  and  coherent,  to  tear  it  away 
from   the   culture   medium.     It   requires   almost   as   much 
skill  to  withdraw  the  wire  from  the  colony  without  touching 
anything  as  to  successfully  approach  the  colony  in  the  first 
place.     The   bacterial  mass  adhering  to  the  wire  is  now 
spread  upon  the  surface  of  agar-agar  or  stabbed  in  gelatin 
or  stirred   in   fluid   medium,    as   the  case   may  be.      The 
higher   the   magnification   under  which   this    operation   is 
done,   the  more  difficult  it  is.     Therefore  only  low-power 
lenses  should  be  employed.     The  transplantation  should  be 
made  immediately  after  the  wire  has  touched  the  colony, 
the  wire  not  being  permitted  in  the  meantime  to  come  into 
contact  with  any  other  object.     Immediately  after  receiving 
the  inoculation,  the  tube  containing  the  culture  medium, 
held  in  the  left  hand,   should  be  passed  with  a  twisting 
motion  through  the  flame  of  a  Bunsen  burner,  so  that  its 
mouth  and  the  cotton  plug  shall  be  heated.     The  cotton 
plug  of  course  takes  fire,  but  by  vigorously  blowing  once  or 
twice  the  fire  can  be  extinguished.     The  cotton  is  then  re- 
moved   with    a  twisting  motion   of    the   plug  and   placed 
between  two  fingers  of  the  left  hand  in  such  a  manner  that 
only  the  extra-tubular  portion  of  the  cotton  is  touched  by 
the  fingers.      During  the  time  that  the  plug  is  being  with- 
drawn the  tube  should  be  held  as  nearly  as  possible  in  a 
horizontal  position,  in  order  that  nothing  may  fall  into  it 
from  the  atmosphere.     The  inoculation  having  been  made, 
the  plug  is  returned  and  the  platinum  wire  heated  to  redness 
in  order  that  any  remaining  bacteria  may  be  killed  by  the 
heat. 


254 


Cultures,  and  their  Study 


B 


The  Gelatin  Puncture  Culture. — To  make  satisfactory 
puncture  cultures,  the  gelatin  must  be  firm  and  not  old  and 
dry.  Should  the  gelatin  be  soft  and  semi-fluid  at  the  time 
the  puncture  is  made,  the  bacteria  diffuse  themselves  through- 
out and  the  typical  appearance  of  the  growth  may  be 
masked.  On  the  other  hand,  if  the  gelatin  is  old,  dry,  or 
retracted,  it  is  very  apt  to  crack  after  the  culture  has  been 


Fig.  60. — Types  of  growth  in  stab  cultures.  A,  Non-liquefying: 
1,  Filiform  (B.  coli) ;  2,  beaded  (Str.  pyogenes) ;  3,  echinate  (Bact. 
acidi-lactici) ;  4,  villous  (Bact.  murisepticum) ;  5,  arborescent  (B. 
mycoides).  B,  Liquefying:  6,  Crateriform  (B.  vulgare,  24  hours); 
7,  napiform  (B.  subtilis,  48  hours) ;  8,  infundibuliform  (B.  prodigiosus)  ; 
9,  saccate,  (Msp.  Finkleri) ;  10,  stratiform  (Ps.  fluorescens)  (Frost). 


made  and  thus  entirely  destroy  the  characteristics  of  the 
growth.  The  wire  used  in  the  operation  should  be  perfectly 
straight,  and  the  puncture  should  be  made  from  the  center 
of  the  surface  directly  down  to  the  bottom  of  the  tube  and 
then  withdrawn,  so  that  a  simple  puncture  is  made.  The 
resulting  appearances  as  the  growth  progresses  are  subject 
to  striking  variations  according  to  the  liquefying  or  non- 


The  Agar-agar  Culture 


255 


liquefying  tendency  of  the  micro-organisms.  Various  types 
of  gelatin  cultures  are  shown  in  the  accompanying  diagrams, 
and  it  is  rather  important  that  the  student  should  familiarize 
himself  with  the  terms  by  which  these  different  growths  are 
described,  in  order  that  uniformity  of  description  may  be 
maintained.  Gelatin  cultures  may  not  be  kept  in  the  incu- 
bating oven,  as  the  medium  liquefies  at  such  temperatures. 
On  the  other  hand,  it  must  not  be  kept  where  the  tempera- 
ture is  too  low,  else  the  bacterial  growth  may  be  retarded. 
The  temperature  of  a  comfortably  heated  room,  not  subject 
to  excessive  variations,  such  as  are  caused  by  steam  heat 
and  the  burning  of  gas,  etc.,  is  about  the  most  appropriate. 
Like  the  colonies,  the  cultures  must  be  carefully  examined 
from  day  to  day,  as  it  not  infrequently  happens  that  a  growth 


Fig.  61.— Types  of  streak  cultures :  1,  Filiform  (B.  coli) ;  2,  echinulate 
(Bact.  acidi-lactici) ;  3,  beaded  (Str.  pyogenes) ;  4,  effuse  (B.  vulgaris) ; 
5,  arborescent  (B.  mycoides)  (Frost). 

which  shows  no  signs  of  liquefaction  to-day  may  begin  to 
liquefy  to-morrow  or  a  week  hence,  or  even  as  late  as  two 
weeks  hence. 

The  Agar=agar  Culture. — Different  operations  vary  in 
the  exact  method  adopted  in  transplanting  to  agar-agar.  In 
most  cases,  a  simple  stroke  is  made  from  the  bottom  of  the 
tube  in  which  the  agar-agar  has  been  obliquely  solidified,  and 
where  it  is  fresh  and  moist,  to  the  upper  part,  where  it  is 
thin  and  dry.  In  addition  to  this,  it  is  advisable  to  make 
a  puncture  from  the  center  of  the  oblique  surface  to  the  bot- 
tom of  the  tube.  This  enables  us  to  tell  whether  the  bacteria 
can  grow  as  readily  below  the  surface  as  above.  Some 
workers  always  make  a  zigzag  stroke  upon  the  surface  of  the 
agar-agar.  This  does  not  seem  to  have  any  particular 


256  Cultures,  and  their  Study 

advantage  except  in  cases  where  it  is  desired  to  scatter  the 
transplanted  organisms  as  much  as  possible,  in  order  that 
the  bacteria  will  grow,  or  in  order  that  a  large  bacterial  mass 
may  be  secured. 

Cultures  upon  Potato. — These  are  made  by  simply 
stroking  the  surface  of  the  culture  medium,  the  opacity  of 
the  potato  making  it  impracticable  to  puncture  it. 

Cultures  in  Fluid  Media. — Here,  as  has  already  been 
stated,  transplantation  consists  in  simply  stirring  in  the 
bacteria  so  as  to  distribute  them  fairly  well  throughout  the 
medium. 

Adhesion  Preparations. — Sometimes  it  is  desirable  to 
preserve  an  entire  colony  as  a  permanent  microscopic  speci- 
men. To  do  this  a  perfectly  clean  cover-glass,  not  too  large 
in  size,  is  momentarily  warmed,  then  carefully  laid  upon  the 
surface  of  the  gelatin  or  agar-agar  containing  the  colonies. 
Sufficient  pressure  is  applied  to  the  surface  of  the  glass  to 
exclude  bubbles,  but  not  to  destroy  the  integrity  of  the 
colony.  The  cover  is  gently  raised  by  one  edge,  and  if  suc- 
cessful the  whole  colony  or  a  number  of  colonies,  as  the  case 
may  be,  will  be  found  adhering  to  it.  It  is  treated  exactly 
as  any  other  cover-glass  preparation — dried,  fixed,  stained, 
mounted,  and  kept  as  a  permanent  specimen.  It  is  called 
an  adhesion  preparation — " Klatschprdparat." 

Special  Methods  of  Securing  Pure  Cultures.— Pure 
cultures  from  single  colonies  may  also  be  secured  by  a  very 
simple  manipulation  suggested  by  Banti.*  The  inoculation 
is  made  into  the  water  of  condensation  at  the  bottom  of  an 
agar-agar  tube,  without  touching  the  surface.  The  tube  is 
then  inclined  so  that  the  water  flows  over  the  agar,  after 
which  it  is  stood  away  in  the  vertical  position.  Colonies  will 
grow  where  bacteria  have  been  floated  upon  the  agar-agar, 
and  may  be  picked  up  later  in  the  same  manner  as  from  a 
plate. 

When  the  bacterium  to  be  isolated  (gonococcus,  etc.) 
will  not  grow  upon  the  media  capable  of  alternate  solidifica- 
tion and  liquefaction,  the  blood-serum,  potato,  or  other 
medium  may  be  repeatedly  stroked  with  the  platinum  wire 
dipped  in  the  material  to  be  investigated.  Where  the  first 
strokes  were  made,  confluent  impure  cultures  occur;  but  as 
the  wire  became  freer  of  organisms  by  repeated  contact  with 
the  medium,  the  colonies  become  scattered  and  can  be 
studied  and  transplanted. 

*  "  Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1895,  xvii,  No.  16. 


Special  Methods  of  Securing  Pure  Cultures     257 

In  some  cases  pure  cultures  may  be  most  satisfactorily 
secured  by  animal  inoculation.  For  example,  when  the 
tubercle  bacillus  is  to  be  isolated  from  milk  or  urine  which 
contains  bacteria  that  would  outgrow  the  slow-developing 
tubercle  bacillus,  it  is  necessary  to  inject  the  fluid  into  the 
abdominal  cavity  of  a  guinea-pig,  await  the  development  of 
tuberculosis  in  the  animal,  and  then  seek  to  secure  pure  cul- 
tures of  the  bacillus  from  the  unmixed  infectious  material  in 
the  softened  lymphatic  glands. 

In  many  cases,  when  it  is  desired  to  isolate  Micrococcus 
tetragenus,  the  pneumococcus,  and  others,  it  is  easier  to 
inoculate  the  animal  most  susceptible  to  the  infection  and 
recover  it  from  the  blood  or  organs,  than  to  plate  it  out  and 
search  for  the  colony  among  many  others  similar  to  it. 

The  growth  upon  agar-agar  is  in  many  ways  less  charac- 
teristic than  in  gelatin,  but  as  the  medium  does  not  liquefy 
except  at  a  high  temperature  (100°  C.),  it  has  that  advan- 
tage. The  colorless  or  almost  colorless  condition  of  the 
preparation  also  aids  in  the  detection  of  chromogenesis. 

Sometimes  the  growth  is  colored ;  at  times  the  production 
of  soluble  pigment  colors  the  agar-agar  as  well  as  the  growth ; 
sometimes  the  bacterial  mass  has  one  color  and  the  agar-agar 
another.  The  growth  may  be  filamentous,  or  simply  a 
smooth,  shining  band.  Occasionally  the  bacterium  does  not 
grow  upon  agar-agar  unless  glycerin  be  added  (tubercle 
bacillus);  sometimes  it  will  not  grow  even  then  (gono- 
coccus). 

Still  less  characteristic  are  the  growths  upon  potato. 
Most  bacteria  produce  smooth,  shining,  irregularly  extending 
growths,  that  may  show  characteristic  colors. 

In  milk  and  litmus  milk  one  should  observe  change  in 
color  from  the  occurrence  of  acid  or  alkali  production, 
coagulation,  gelatinization,  and  digestion  of  the  coagulum. 

Blood-serum  is  liquefied  by  some  bacteria,  but  the  major- 
ity of  organisms  have  no  characteristic  reaction  upon  it.  A 
few,  as  the  bacillus  of  diphtheria,  are,  however,  characterized 
by  rapid  development  at  given  temperatures. 

While  most  of  the  saprophytic  bacteria  grow  well  at  the 
temperature  of  *a  well-wTarmed  room,  the  important  patho- 
genic forms  must  be  kept  at  the  temperature  of  the  body 
either  to  permit  growth  or  to  secure  typical  development. 
To  do  this  satisfactorily  an  incubating  oven  or  thermostat  be- 
comes a  necessity.  Various  forms,  of  wood  and  metal,  are  in 
the  market,  one  being  shown  in  the  illustration  (Fig.  62). 
17 


258 


Cultures,  and  their  Study 


The  growth  in  gelatin  is  generally  so  far  removed  from 
the  walls  of  the  tube  (a  central  puncture  nearly  always  being 


Fig.  62. — Modern  incubating  oven. 

made  in  the  culture-medium,  in  order  that  the  growth  be 
symmetric)  that  it  is  impossible  to  make  a  microscopic  ex- 


Microscopic  Study  of  Cultures  259 

amination  of  it  with  any  power  beyond  that  given  by  a 
hand -lens. 

MICROSCOPIC  STUDY  OF  CULTURES. 

Some  attention  has  been  given  to  the  preparation  of  micro- 
tome sections  of  the  gelatin  growth,  which  can  be  done  if  the 
glass  be  warmed  just  sufficiently  to  permit  the  gelatin  con- 
taining the  growth  to  be  removed  and  placed  in  Miiller's 
fluid  (bichromate  of  potassium  2-2.5,  sulphate  of  sodium  i, 
water  100),  where  it  is  hardened.  When  quite  firm  it  is 
washed  in  water,  passed  through  alcohols  ascending  in 
strength  from  50  to  100  per  cent.,  embedded  in  celloidin,  cut 
wet,  and  stained  like  a  section  of  tissue. 

A  ready  method  of  doing  this  has  been  suggested  by 
Winkler,*  who  bores  a  hole  in  a  block  of  paraffin  with  the 
smallest  size  cork-borer,  soaks  the  block  in  bichlorid  solution 
for  an  hour,  pours  liquid  gelatin  into  the  cavity,  allows  it  to 
solidify,  inoculates  it  by  the  customary  puncture  of  the 
platinum  wire,  allows  it  to  develop  sufficiently,  and  when 
ready  cuts  the  sections  under  alcohol,  subsequently  staining 
them  with  much  diluted  carbol-fuchsin. 

Neat  museum  specimens  of  plate  and  puncture  cultures 
in  gelatin  can  be  made  by  simultaneously  killing  the  micro- 
organisms and  permanently  fixing  the  gelatin  with  formal- 
dehyd,  which  can  either  be  sprayed  upon  the  gelatin  or  ap- 
plied in  dilute  solution.  As  gelatin  fixed  in  formaldehyd 
cannot  subsequently  be  liquefied,  such  preparations  will  last 
indefinitely. 

Standardizing  Freshly  Isolated  Cultures. — This  is  a 
matter  of  some  importance,  as  in  bringing  bacteria  into 
the  new  environment  of  artificial  cultivation  their  bio- 
logic peculiarities  are  temporarily  altered,  and  it  takes 
some  time  for  them  to  recover  themselves.  While  the 
appearances  of  the  freshly  isolated  organism  should  be 
carefully  noted,  too  much  stress  should  not  be  laid  upon 
them,  and  before  beginning  the  systematic  study  of  any 
new  organism  it  should  be  made  to  grow  for  several  suc- 
cessive generations  upon  two  or  three  of  the  most  impor- 
tant culture  media.  Its  saprophytic  existence  being  thus 
established,  the  characteristics  manifested  become  the  per- 
manent peculiarities  of  the  species. 

*  "  Fortschritte  der  Medicin,"  Bd.  xi,  1893,  No.  22. 


CHAPTER  IX. 

THE  CULTIVATION  OF  ANAEROBIC  ORGANISMS. 

THE  presence  of  uncombined  oxygen  in  ordinary  cul- 
tures inhibits  the  development  of  anaerobic  bacteria. 
When  such  are  to  be  cultivated,  it  therefore  becomes 
necessary  to  utilize  special  apparatus  or  adopt  physical 
or  chemic  methods  for  the  exclusion  of  the  air.  Many 
methods  have  been  suggested  for  the  purpose,  an  excel- 
lent review  of  which  has  recently  been  published  by  Hun- 
ziker,*  who  divides  them  as  follows,  according  to  the 
principle  by  which  the  anaerobiosis  is  brought  about : 

1.  By  the  formation  of  a  vacuum. 

2.  By  the  displacement  of  the  air  by  inert  gases. 

3.  By  the  absorption  of  the  oxygen. 

4.  By  the  reduction  of  the  oxygen. 

5.  By  the   exclusion  of    atmospheric   air  by  means  of 

various  physical  principles  and  mechanical  devices. 

6.  By  the  combined  application  of  any  two  or  more  of 

the  above  principles. 

This  classification  makes  such  an  excellent  foundation  for 
the  description  of  the  methods  that  it  has  been  unhesitatingly 
adopted. 

1.  Withdrawal  of  the  Air  and  the  Formation  of  a 
Vacuum. — This  method  was  first  suggested  by  Pasteur  and 
was  later  modified  by  Roux,  Gruber,  Zupinski,  Novy,  and 
others.     It  is  now  very  rarely  employed.     The  appropriate 
container,  whether  a  tube,  flask,  or  some  special  device  such 
as  the  Novy  jar  (Fig.  63),  receives  the  culture,  and  then  has 
the  air  removed  by  a  vacuum  pump,  the  tube  either  being 
sealed  in  a  flame  or  closed  by  a  stop-cock  to  prevent  the  re- 
entrance  of  the  air 

2.  Displacement   of   the   Air   by    Inert   Gases. — This 
method   is    decidedly  preferable    to    the    preceding,   as    it 

*  "Journal  of  Applied  Microscopy  and  Laboratory  Methods,"  March, 
April,  and  May,  1902;  vol.  v,  Nos.  3,  4,  and  5. 

260 


Displacement  of  the  Air  by  Inert  Gases      261 

leaves  no  vacuum.  It  is  easier  to  displace  the  oxygen  than 
to  withdraw  it,  and  any  apparatus  permitting  a  combina- 
tion of  both  features,  as  that  designed  by  Ravenel,*  from 
which  the  air  can  be  sucked  by  a  pump,  to  be  later  replaced 
by  hydrogen,  can  be  viewed  with  favor. 

The  most  simple  apparatus  of  the  kind  was  suggested  by 
Frankel  (Fig.  64),  who  inoculated  a  culture-tube  of  melted 
gelatin  or  agar-agar,  solidified  it  upon  the  wall  of  the  tube,  as 
suggested  by  Esmarch,  substituted  for  the  cotton  stopper  a 
sterile  rubber  cork  containing  a  long  entrance  and  short 
exit  tube  of  glass,  passed  hydrogen  through  the  tube  until 
the  oxygen  had  been  entirely  removed,  then  sealed  the 
ends  in  a  flame.  In  this  tube  the  growth  of  superficial  and 
deep  colonies  can  be  observed.  Hansen  and  Liborius  con- 


Fig.  63. — Novy's  jars  for  anaerobic  cultures. 

structed  special  tubes  (Fig.  65)  by  fusing  a  small  glass  tube 
into  the  wall  of  a  culture-tube,  and  narrowing  the  upper  part 
of  the  tube  in  a  flame.  After  inoculation,  hydrogen  is  passed 
into  the  small  tube  and  permitted  to  escape  through  the 
mouth  of  the  large  tube  until  the  air  is  entirely  replaced, 
after  which  both  tubes  are  sealed  in  a  flame. 

Instead  of  having  a  special  apparatus  for  each  culture,  it 
is  far  better  to  adapt  the  principle  to  some  larger  piece  of 
apparatus  that  can  contain  a  number  of  tubes  or  Petri  dishes 
at  a  time.  For  this  purpose  the  jar  invented  by  Novy  or  the 
apparatus  of  Botkin  can  be  used. 

The  Novy  jar  receives  as  many  inoculated  tubes  as  it  will 

*" Bacteria  of  the  Soil,"  "Memoirs  of  the  National  Academy  of 
Sciences,"  First  Memoir,  1896. 


262     The  Cultivation  of  Anaerobic  Organisms 

contain  and  has  its  stopper  so  replaced  that  the  openings  in 
the  neck  and  stopper  correspond.  Hydrogen  gas  is  passed 
through  until  the  air  is  displaced.  This  usually  takes  sev- 
eral hours,  as  the  cotton  stoppers  retain  the  air  in  the  test- 
tubes  and  prevent  rapid  diffusion.  When  the  air  is  all  dis- 
placed, the  stopper  is  turned  so  that  the  tubes  are  closed. 
If  it  be  desired  to  expedite  matters  a  pump  can  be  used  to 
withdraw  the  air,  after  which  the  hydrogen  is  permitted  to 
enter. 

Botkin's  apparatus  is  intended  for  cultures  in  Petri  dishes. 


Cotton  jtLvf 


Fig.   64.— Frankel's  method  of 
making  anaerobic  cultures. 


Fig.  65. — Liborius'  tube  for 
anaerobic  cultures. 


It  consists  of  three  parts — a  deep  dish  of  glass  (6),  a  stand 
to  support  the  Petri  dishes  to  be  exposed  (c),  and  a  bell- 
glass  (a)  to  cover  the  stand  and  fit  inside  of  the  dish.  It 
can  easily  be  understood  by  reference  to  figure  66.  The 
prepared  dishes  are  stood  uncovered  in  the  rack,  which  is 
then  placed  in  the  dish  forming  the  bottom  of  the  appara- 
tus, and  into  which  liquid  paraffin  is  poured  to  a  depth  of 
about  two  inches.  The  bell-glass  cover  is  now  stood  in 
place  and  hydrogen  gas  is  conducted  through  previously 
arranged  rubber  tubes  ( d,  e) .  As  soon  as  the  air  is  displaced 


The  Absorption  of  the  Atmospheric  Oxygen     263 

through  tube  d,  both  tubes  are  withdrawn.  It  is  well  to 
place  one  Petri  dish  containing  alkaline  pyrogallic  acid  in 
the  rack  to  absorb  any  oxygen  not  successfully  displaced. 

3.  The  Absorption  of  the  Atmospheric  Oxygen. This 

method  was  first  suggested  by  Buchner,  whose  idea  was  to 
absorb  the  atmospheric  oxygen  by  alkaline  pyrogallic  acid 
and  permit  the  bacteria  to  develop  in  the  indifferent  nitro- 
gen. Various  methods  have  been  suggested  for  achieving 
this  end,  Buchner's  own  method  consisting  in  the  use  of  two 
tubes,  a  small  one  to  contain  the  culture  (Fig.  68)  and  a  larger 
one  to  contain  the  absorbing  fluid.  A  fresh  solution  of  pyro- 
gallic acid  and  sodium  hy- 
droxid  were  poured  into 
the  large  tube,  the  smaller 
tube  placed  within  it,  upon 
some  appropriate  sup- 
port, and  the  whole  tightly 
corked. 

Nichols  and  Schmitter,* 
at  the  suggestion  of  Carroll, 
have  modified  the  method 
by  connecting  the  tube  con- 
taining the  inoculated  cul- 
ture medium  with  a  U- 
shaped  tube,  to  the  other 
end  of  which  is  attached 
a  tube  to  contain  the  pyro- 
gallic acid  solution.  The 
apparatus  will  at  once  be 
understood  by  a  glance  at  ] 
the  cut  (Fig.  67).  The  mode 
of  employing  it  is  as  follows :  ' '  After  inoculating  the  culture- 
tube  the  plug  is  pushed  in  a  little  below  the  lips  of  the  tube ; 
the  ends  of  the  U  tube  and  the  test-tubes  are  coated  exter- 
nally with  vaselin,  the  rubber  tubes  are  adjusted  on  the 
U  tube  and  a  connection  made  with  the  culture-tube  so  that 
the  glass  ends  meet.  One  or  two  grams  of  pyrogallic  acid 
are  put  in  the  empty  test-tube  and  packed  down  with  a 
little  filter-paper  over  it;  ten  or  twenty  cubic  centimeters, 
respectively,  of  a  10  per  cent,  solution  of  sodium  hydroxide 
are  then  poured  into  the  tube  and  the  second  connection 
made  before  the  acid  and  alkali  react  to  any  extent." 
*  "Jour,  of  Medical  Research,"  July,  1906,  p.  113. 


66. — Bodkin's   apparatus    for 
making  anaerobic  cultures. 


264     The  Cultivation  of  Anaerobic  Organisms 


Wright  has  given  the  most  simple  modification  by  sug- 
gesting that  the  cotton  stopper  of  the  ordinary  culture-tube 

have  its  projecting  part  cut  off 
and  the  plug  itself  pushed  down 
the  tube  for  a  short  distance. 
Some  alkaline  pyrogallic  acid  so- 
lution is  poured  upon  the  cotton, 
to  saturate  it,  and  the  tube 
tightly  corked. 

Zinsser*  has  also  adopted  the 
method  so  as  to  be  very  satis- 
factory for  use  with  Petri  dishes. 
The  dishes  selected  should  be 
rather  deeper  than  ordinary. 
They  are  sterilized  and  inocu- 
lated in  the  ordinary  manner  and 
then  inverted.  The  dish  is  cau- 
tiously raised,  and  some  pyro- 
gallic acid  carefully  poured  into 
the  lid  and  the  dish  gently 
dropped  into  place  again.  The 
alkaline  solution  is  then  poured 
into  the  crevice  between  the 
edges  of  the  dish  and  the  lid, 
and  then  the  remainder  of  the 
space  filled  with  melted  albolene. 
When  these  dishes  are  carefully 
stood  away,  the  alkaline  pyro- 
gallic acid  absorbs  all  of  the  con- 
tained oxygen  and  the  anaerobic 
cultures  develop  quite  well.  The 
growing  colonies  can  be  exam- 
ined as  often  as  may  be  necessary 

through  the  bottom  of  the  dishes,  which  must,  of  course, 
always  be  kept  in  the  inverted  position. 

4.  Reduction    of   Oxygen. — Pasteur   and,    later,    Roux 
have  recommended  the  cultivation  of  anaerobic  bacteria  in 
association  with  aerobic  bacteria  by  which  the  oxygen  was 
to  be  absorbed.     This  method  is  too  crude  to  be  employed 
at  the  present  time,  as  it  destroys  the  essential  character- 
istics of  the  cultures  by  mixing  the  products  of  the  bacteria. 
Chemic  reduction  of  the  oxygen  has  been  attempted  by 
the  addition  of  2  per  cent,  of  glucose,  as  suggested  by  Libo- 
*  "Journal  of  Experimental  Medicine,"  1906,  vin,  542. 


Fig.  67. —  Spirillum  ru- 
brum.  Glucose  agar  slant 
culture  of  five  days.  Abun- 
dant production  of  pigment 
on  the  surface.  (The  U  tube 
was  soiled  by  the  reducing 
fluid  during  handling  by  the 
photographer.)  (Nichols  and 
Schmitter.) 


.    Exclusion  of  Atmospheric  Oxygen  265 

rius,  0.3-0.5  per  cent,  of  sodium  formate,  as  suggested  by 
Kitasato  and  Weil,  o.i  per  cent,  of  sodium  sulphate,  sug- 
gested by  the  same  authors,  and  various  other  chemicals. 
None  of  these  additions  has  been  sufficiently  successful  to 
merit  continued  favor,  and  at  the  present  time  this  method 
is  not  employed. 

5.  Exclusion  of  Atmospheric  Oxygen  by  Means  of 
Various  Physical  Principles  and  Mechanical  Devices.— 
This  has  appealed  to  the  ingenuity  of  many  experimenters, 
and  many  means  of  accomplishing  the  atmospheric  exclusion 
have  been  tried  with  success. 

The  most  simple  plan  is  that  of  Hesse,  who  made  a  deep 
puncture  in  recently  boiled  and  rapidly  cooled  gelatin  or 
agar-agar,  then  covered  the  surface  of  the  medium  with 
sterile  oil  (Fig.  69).  The  so-called  "shake  culture"  is  an- 


Fig.    68. — Buchner's    method    of 
making  anaerobic  cultures. 


Fig.  69. — Hesse's  method  of  mak- 
ing anaerobic  cultures. 


other  very  simple  method,  suggested  by  Liborius  and  Hesse. 
The  medium  to  be  inoculated,  contained  in  a  well-filled  tube 
or  flask,  is  boiled  to  displace  the  contained  air,  cooled  so  as 
no  longer  to  endanger  the  introduced  bacteria,  then  inocu- 
lated, the  inoculated  bacteria  being  distributed  by  gently 
shaking.  On  cooling,  the  medium  "sets,"  the  organisms 
below  the  surface  remaining  under  anaerobic  conditions. 


266     The  Cultivation  of  Anaerobic  Organisms 


Kitasato  first  used  paraffin  as  a  covering  for  the  inoculated 
medium,  his  recommendation  having  recently  been  revived 
and  made  successful  use  of  by  Park  for  the  cultivation  of  the 
tetanus  bacillus.  The  paraffin  floats  upon  the  surface  of  the 
medium,  melts  during  sterilization,  but  does  not  mix  with 
it,  and  "sets"  when  cool.  The  inoculation  is  to  be  made 
while  the  culture  medium  is  warm,  after  boiling  and  before 
the  paraffin  sets. 

Koch  studied  the  colonies  of  anaerobic  organisms  by  culti- 
vating them  upon  a  film  of  gelatin 
covered  by  a  thin  sheet  of  steril- 
ized mica,  by  which  the  air  was 
excluded. 

Salomonsen  has  made  use  of  a 
pipet  for  making  anaerobic  cul- 
tures. It  is  made  of  a  glass  tube 
a .  few  millimeters  in  diameter, 
drawn  out  to  a  point  at  each 
end.  The  inoculated  gelatin  or 
agar-agar  is  drawn  in  while  lique- 
fied and  the  ends  sealed.  The 
tube,  of  course,  contains  no  air, 
and  perfect  anaerobiosis  results. 

Theobald  Smith  has  found  the 
fermentation-tube  and  various 
modifications  of  it  excellently  well 
adapted  to  the  growth  of  anae- 
robes, which,  of  course,  grow  only 
in  the  closed  limb. 

Hens'  eggs  have  been  used  for 
anaerobic  cultures,  and  in  them 
the  tetanus  bacillus  grows  remark- 
ably well.  Conditions  of  anaero- 
biosis are,  however,  not  perfect, 
as  can  be  shown  by  the  behavior 
of  the  egg  itself.  If  oxygen  be 
completely  shut  out  by  oiling  or 
varnishing  the  shell,  a  fertile  egg 
will  not  develop. 

A  quite  satisfactory  and  simple 
device  for  routine  work  with 

anaerobic  organisms  has  been  invented  by  Wright  *  (Figs. 

70  and  71).     The  essential  feature  consists  of  a  pipet,  D, 

*  "Jour.  Boston  Soc.  of  Med.  Sci.,"  Jan.,  1900. 


Figs.  70,  71.— Wright's 
method  of  making  anaero- 
bic cultures  in  fluid  media 
(Mallory  and  Wright). 


Exclusion  of  Atmospheric  Oxygen  267 

with  a  rubber  tube,  E,  at  the  end,  and  one  interruption 
connected  by  a  rubber  tube,  C.  The  device  will  be  made 
clear  at  once  by  a  glance  at  the  accompanying  illustration. 
The  method  of  employment  is  very  simple.  An  ordinary 
tube  of  bouillon  or  other  fluid  culture  media  receives  the 
pipet,  the  whole  being  sterilized,  the  cotton  plug  in  place. 
The  bouillon  being  inoculated  with  the  culture  or  secretion 
to  be  studied  is  drawn  up  in  the  bulb  of  the  pipet,  A,  by 
suction,  until  it  passes  the  rubber  interruption,  C.  By 
forcing  the  upper  end  of  the  pipet  downward  in  the  test-tube, 
a  kink  is  given  each  rubber  tube  and  the  fluid  contained  in 
the  bulbous  part  of  the  pipet  becomes  hermetically  sealed. 

In  all  cases  where  the  presence  of  suspected  micro- 
organisms is  to  be  demonstrated,  it  is  necessary  to  make 
both  aerobic  and  anaerobic  cultures.  For  routine  work  of 
this  kind,  this  method  of  Wright  is  probably  the  most  con- 
venient yet  suggested. 


CHAPTER  X. 
EXPERIMENTATION  UPON  ANIMALS* 

THE  principal  objects  of  medical  bacteriology  are  to  dis- 
cover the  cause,  explain  the  symptoms,  and  bring  about  the 
cure  and  future  prevention  of  disease.  We  cannot  hope 
to  achieve  these  objects  without  experimentation  upon 
animals,  in  whose  bodies  the  effects  of  bacteria  and  their 
products  can  be  studied. 

No  one  should  more  heartily  condemn  wanton  cruelty  to 
animals  than  the  physician.  Indeed,  it  is  hard  to  imagine 
men,  so  much  of  whose  life  is  spent  in  relieving  pain,  and 
who  know  so  much  about  pain,  being  guilty  of  the  butchery 
and  torture  accredited  to  them  by  a  few  of  the  laity,  whose 
eyes,  but  not  whose  brains,  have  looked  over  the  pages 
of  physiologic  text-books,  and  whose  "philanthropy  has 
thereby  been  transformed  to  zoolatry." 

It  is  entirely  through  experimentation  upon  animals  that 
we  have  attained  our  knowledge  of  physiology,  most  of 
our  important  knowledge  of  therapeutics,  and  most  of  our 
knowledge  of  the  infectious  diseases.  Without  its  aid  we 
would  still  be  without  one  of  the  greatest  achievements  of 
medicine,  the  serum  therapy  of  diphtheria. 

Experiments  upon  animals,  therefore,  must  be  made,  and, 
as  the  lower  animals  differ  in  their  susceptibility  to  diseases, 
large  numbers  and  different  kinds  of  animals  must  be  em- 
ployed. 

The  bacteriologic  methods  are  fortunately  not  cruel,  the 
principal  modes  of  introducing  bacteria  into  the  body  being 
by  subcutaneous,  intraperitoneal,  and  intravenous  injec- 
tion. 

Any  hypodermic  syringe  that  can  conveniently  be  cleaned 
and  disinfected  may  be  employed  for  the  purpose.  Forms 
expressly  designed  for  bacteriologic  work  and  most  fre- 

268 


Animal  Inoculations 


269 


quently  employed  are  shown  in  figure  72.     Those  of  Meyer 
and  Roux  resemble  ordinary  hypodermic  syringes;  that  of 


Fig.  72. — i.  Roux's  bacteriologic  syringe;  2,  Koch's  syringe;  3,  Meyer's 
bacteriologic  syringe. 


ig-  73- — Altmann  syringes  for  bacteriologic  and  hematologic  work. 


Koch  is  supposed  to  possess  the  decided  advantage  of  not 
having  a  piston  to   come   into  contact  with  the   fluid  to 


270  Experimentation  upon  Animals 

be  injected.  This  is,  however,  really  disadvantageous, 
inasmuch  as  the  cushion  of  compressed  air  that  drives  out 
the  contents  is  elastic,  and  unless  carefully  watched  will 
follow  the  injection  into  the  body  of  the  animal.  In  making 
subcutaneous  injections  there  is  no  disadvantage  or  danger 
from  the  entrance  of  air,  but  in  intravenous  injections  it  is 
extremely  dangerous. 

Syringes  with  metal  or  glass  pistons  are  excellent,  though 
not  very  durable.  All  syringes  should  be  disinfected  with  5 
per  cent,  carbolic  acid  solutions  before  and  after  using,  the 
carbolic  acid  being  allowed  to  act  for  some  time  and  then 
washed  out  with  sterile  water.  Syringes  should  not  often  be 


Fig.  74. — Method  of  making  an  intravenous  injection  into  a  rabbit. 
Observe  that  the  needle  enters  the  posterior  vein  from  the  hairy 
surface. 


boiled,  as  it  ruins  the  packings,  whether  of  asbestos,  leather, 
or  rubber. 

The  intravenous  injections  differ  only  in  that  the  needle 
of  the  syringe  is  introduced  into  a  vein.  This  is  easy  to 
achieve  in  a  large  animal,  like  a  horse,  but  is  very  difficult 
in  a  small  animal,  and  well-nigh  impossible  in  anything 
smaller  than  a  rabbit.  Such  injections,  when  given  to  rab- 
bits, are  usually  made  into  the  ear-veins,  which  are  most 


Animal  Inoculations  271 

conspicuous  and  accessible  (Fig.  74).  A  peculiar  and  im- 
portant fact  to  remember  is  that  the  less  conspicuous  poste- 
rior -vein  of  the  ear  is  much  better  adapted  to  the  purpose 
than  the  anterior.  The  introduction  of  the  needle  should  be 
made  from  the  hairy  external  surface  of  the  ear  when  the 
vein  is  immediately  beneath  the  skin. 

If  the  ear  be  manipulated  for  a  moment  or  two  before  the 
injection,  vasomotor  dilatation  occurs  and  the  blood-vessels 
become  larger  and  more  conspicuous.  The  vein  should  be 
compressed  at  the  root  of  the  ear  until  the  needle  is  intro- 
duced, and  the  injection  made  as  near  the  root  as  possible. 
The  fluid  should  be  slowly  injected. 

Bacteria  can  be  introduced  into  the  lymphatics  only  by 
injecting  liquid  cultures  of  them  into  some  organ  with  com- 
paratively few  blood-vessels  and  large  numbers  of  lym- 
phatics. The  testicle  is  best  adapted  to  this  purpose,  the 
needle  being  introduced  deeply  into  the  organ. 

Sometimes  subcutaneous  inoculations  are  made  by  intro- 
ducing the  platinum  wire  through  a  small  opening  made  in 
the  skin  by  a  snip  of  the  scissors.  By  this  means  solid 
cultures  from  agar-agar,  etc.,  can  be  introduced. 

Intra-abdominal  and  intrapleural  injections  are  some- 
times made,  and  in  cases  where  it  becomes  necessary  to 
determine  the  presence  or  absence  of  the  bacilli  of  tuber- 
culosis or  glanders  in  fragments  of  tissue  it  may  be  neces- 
sary to  introduce  small  pieces  of  the  suspected  tissue 
under  the  skin.  To  do  this  the  hair  is  closely  cut  over 
the  point  of  election,  which  is  generally  on  the  abdo- 
men near  the  groin,  the  skin  picked  up  with  forceps,  a 
snip  made  through  it,  and  the  points  of  the  scissors  intro- 
duced for  an  inch  or  so  and  then  separated.  By  this  man- 
oeuver  a  subcutaneous  pocket  is  formed,  into  which  the 
tissue  is  easily  forced.  The  opening  should  not  be  large 
enough  to  require  subsequent  stitching. 

When  tissue  fragments  or  collodion  capsules  are  to  be  in- 
troduced into  the  abdominal  cavity,  the  animal  should  be 
anesthetized  and  a  formal  laparotomy  done,  the  wound  being 
carefully  stitched  together. 

When,  in  studying  Pfeiffer's  phenomenon  and  similar  con- 
ditions, it  is  desirable  occasionally  to  withdraw  drops  of 
fluid  from  the  abdominal  cavity,  a  small  opening  can  be 
burned  through  with  a  blunt  needle.  This  does  not  heal 


272  Experimentation  upon  Animals 


Fig.  75- — Latapie's  animal  holder  for  rabbits,  guinea-pigs,  and  other 
small  animals.  This  form  of  holder  is  in  general  use  at  the  Institute 
Pasteur  in  Paris. 


Fig.  76. — Guinea-pig  confined  in  the  holder. 


Fig.  77. — Mouse-holder. 


Securing  Blood  from  Animals  273 

readily,  and  through  it,  from  time  to  time,  a  capillary  pipet 
can  be  introduced  and  the  fluid  withdrawn. 

Small  animals,  such  as  rabbits  and  guinea-pigs,  can  be 
held  in  the  hand,  as  a  rule.  Guinea-pig  and  rabbit-holders 
of  various  forms  can  be  obtained  from  dealers  in  laboratory 
supplies.  The  best  of  these  is  undoubtedly  that  of  Latapie, 
shown  in  the  accompanying  illustration  (Fig.  75).  Dogs, 
cats,  sheep,  and  goats  can  be  tied  and  held  in  troughs.  A 
convenient  form  of  mouse-holder,  invented  by  Kitasato,  is 
shown  in  figure  77. 

In  all  these  experiments  one  must  remember  that  the 
amount  of  material  introduced  into  the  animal  must  be  in 
proportion  to  its  size,  and  that  injection  experiments  upon 
mice  are  usually  so  crude  and  destructive  as  to  warrant  the 
comparison  drawn  by  Frankel,  that  the  injection  of  a  few 
minims  of  liquid  into  the  pleural  cavity  of  a  mouse  is  "  much 
the  same  as  if  one  would  inject  through  a  fire-hose  three  or 
four  quarts  of  some  liquid  into  the  respiratory  organs  of  a 
man." 

Method  of  Securing  Blood  from  Animals. — For  many 
experimental  purposes  it  becomes  necessary  to  secure  blood 
in  larger  or  smaller  quantities  from  animals.  For  horses, 
cattle,  calves,  goats,  sheep,  large  dogs,  etc.,  this  is  a  simple 
matter,  all  that  is  necessary  being  to  restrain  the  animal, 
make  a  minute  incision  in  the  skin  over  the  jugular  vein, 
which  is  easily  found  by  compressing  it  at  the  root  of  the 
neck  and  noting  where  the  vessel  expands,  and  introducing 
a  canula  when  the  vein  is  well  distended.  The  trocar  being 
withdrawn,  the  blood  at  once  flows.  A  sterile  tube  is 
slipped  over  the  cannula  and  the  blood  conducted  into  a 
sterile  bottle  or  flask. 

For  rabbits  and  guinea-pigs  the  technic  is  rather  more 
difficult  because  of  the  smaller  size  of  the  vessels.  Drops 
and  small  quantities  of  blood  may  be  secured  by  opening 
one  of  the  ear  veins,  but  when  any  quantity  of  blood  is 
required,  the  neatest  operation  is  done  by  tapping  the 
common  carotid  artery  by  the  method  employed  at  the 
Pasteur  Institute  at  Paris. 

The  animal  is  restrained  in  a  Latapie  holder,  with  the 
neck  extended.  Anesthesia  can  be  used,  but  must  be  em- 
ployed with  great  care.  The  hair  on  the  front  of  the  neck 
is  clipped  and  the  neck  shaved,  or,  as  is  easier,  the  hair  is 
18 


274 


Experimentation  upon  Animals 


pulled  out,  -leaving  a  clean  surface  an  inch  square.  The 
skin  is  then  washed  with  a  disinfecting  solution,  an  incision 
one  and  a  half  inches  long  made  through  the  skin  and 
superficial  fascia  in  the  middle  line  of  the  neck,  the  tissues 
carefully  separated,  the  deep  fascia  cautiously  opened,  the 
tissues  separated  with  the  point  of  the  forceps  and  a 
grooved  director,  the  sheath  of  the  ves- 
sels opened,  and  the  artery  completely 
separated  from  its  surrounding  tissues 
for  a  distance  of  at  least  an  inch.  A 
ligature  is  now  tightly  tied  about  the 
artery  at  the  distal  end  of  exposure,  and 
a  ligature  placed  in  position  and  loosely 
looped  ready  to  tie  about  the  proximal 
end.  A  tube  with  a  sharp  lateral  tubu- 
lature,  as  is  shown  in  the  illustration 
(Fig.  78),  is  now  made  ready  by  break- 
ing off  the  closed  tip,  the  moistened 
forefinger  of  the  operator  is  placed  be- 
neath the  artery,  and  the  sharp  tube 
inserted  (point  toward  the  heart)  into 
the  artery,  through  whose  walls  it  cuts 
its  way  easily.  The  moment  the  vessel 
is  entered  the  blood-pressure  drives  the 
blood  into  the  tube  so  that  20  c.c.  may 
be  collected  in  about  as  many  seconds. 
An  assistant  now  ties  the  artery  at  its 
proximal  end,  the  tube  is  withdrawn, 

.-  7f-— Tube  for  holding  it  so  that  the  blood  does  not 
taking  blood  from  the  ,  . 

carotid  artery  of  a  escape,  and  the  end  sealed  in  a  name, 
rabbit  or  guinea-pig.  The  ends  of  the  ligatures  are  now  cut 
short  and  the  external  wound  stitched. 
The  wound  usually  heals  at  once,  and  if  subsequent  study  of 
the  blood  is  required,  the  other  carotid  and  the  femorals 
can  be  similarly  employed  for  obtaining  it. 

Small  quantities  of  blood  (drops)  can  be  secured  from 
mice  and  rats  by  cutting  off  the  tip  of  the  tail,  but  to 
secure  a  large  quantity  is  difficult.  One  method  that  has 
been  recommended  is  to  tie  the  animal  to  a  tray  or  board, 
on  its  back,  anesthetize  it,  and,  just  before  it  dies,  quickly 
open  the  thoracic  cavity,  and  cut  through  the  heart  with 
scissors.  The  animal  at  once  dies,  the  blood  pouring  out 


Post-mortems  upon  Animals  275 

into  the  pleural  cavities.     After  coagulation  the  serum  can 
be  secured  by  carefully  pipetting  it  from  the  cavities. 


-  79- — Showing  the  method  of  taking  blood  from  the  carotid  artery 
of  a  rabbit. 


Post=mortems. — Observation  of  experiment  animals  by 
no  means  ceases  with  their  death.  Indeed,  he  cannot  be  a 
bacteriologist  who  is  not  already  a  good  pathologist  and  ex- 
pert in  the  recognition  of  diseased  organs. 

When  an  autopsy  is  to  be  made  upon  a  small  animal,  it  is 
best  to  wash  it  for  a  few  moments  in  a  disinfecting  solution, 
to  kill  the  germs  present  upon  the  hair  and  skin,  as  well  as 
to  moisten  the  hair,  which  can  then  be  much  more  easily 
kept  out  of  the  incision. 

Small  animals  can  be  tacked  to  a  board  or  tied,  by  cords 
fastened  to  the  legs,  to  hooks  soldered  to  the  corners  of  an 
easily  disinfected  tray.  The  dissection  should  be  made  with 
sterile  instruments.  When  a  culture  is  to  be  made  from  the 
interior  of  an  organ,  its  surface  should  first  be  seared  with  a 
hot  iron,  a  puncture  made  into  it  with  a  sterile  knife,  and 
the  culture  made  by  introducing  a  platinum  wire. 


276  Experimentation  upon  Animals 

If  the  bacteriologic  examination  cannot  be  made  at  once, 
the  organs  to  be  studied  should  be  removed  with  aseptic  pre- 
cautions, wrapped  in  a  sterile  towel  or  a  towel  wet  with  a 
disinfecting  solution,  and  carried  to  the  laboratory,  where 
the  surface  is  seared  and  the  necessary  incisions  made  with 
sterile  instruments. 

Fragments  intended  for  subsequent  microscopic  examina- 
tion should  be  cut  into  small  cubes  (of  i  c.c.)  and  fixed  in 
Zenker's  fluid  or  absolute  alcohol.  (See  page  179.) 

Collodion  capsules  are  quite  frequently  employed  for  the 
purpose  of  cultivating  bacteria  in  a  confined  position  in  the 
body  of  an  animal,  where  they  can  freely  receive  and  utilize 
the  body- juices  without  being  subjected  to  the  action  of  the 
phagocytes.  In  such  capsules  the  bacteria  usually  grow 
plentifully,  and  not  rarely  have  their  virulence  increased. 

The  capsules  can  be  made  of  any  size,  though  they  are 
probably  most  easily  handled  when  of  about  5-10  c.c.  capac- 
ity. The  size  is  always  an  objection,  because  of  the  dis- 
turbance occasioned  when  they  are  introduced  into  the 
abdominal  cavity. 

The  capsules  are  made  by  carefully  coating  the  outside  of 
the  lower  part  of  a  test-tube  with  collodion  until  a  suffi- 
ciently thick,  homogeneous  layer  is  formed.  During  the 
coating  process  the  tube  must  be  twirled  alternately  within 
and  without  the  collodion,  so  that  it  is  equally  distributed 
upon  its  surface.  When  the  desired  thickness  is  attained, 
and  the  collodion  is  sufficiently  firm,  the  tube  is  plunged 
under  water  and  the  hardening  process  checked. 

A  cut  is  next  made  around  the  upper  edge  of  the  collodion: 
film,  and  it  is  removed  by  carefully  turning  it  inside  out.  In 
this  manner  an  exact  mold  of  the  tube  is  formed.  If  a  small 
opening  be  made  at  the  end  of  the  tube  over  which  the  sac  is 
molded,  and  the  tube  filled  with  water  after  being  properly 
coated  with  collodion,  a  small  amount  of  pressure,  applied 
by  blowing  gently  into  the  tube,  will  force  the  water  between 
the  collodion  and  glass  and  so  detach  it  without  inversion. 
A  test-tube  of  the  same  size  is  next  constricted  to  a  degree 
that  will  not  interfere  with  the  future  introduction  of  culture 
media  in  a  fine  pipet  or  inoculation  with  a  platinum  loop, 
and  that  will  permit  of  ready  sealing  in  a  flame  when  neces- 
sary ;  the  rounded  end  is  cut  off,  and  the  edges  are  smoothed 
in  a  flame.  The  upper  open  end  of  the  collodion  bag  is  care- 
fully fitted  over  the  end  of  the  tube,  shrunk  on  by  a  gentle 


Collodion  Capsules 


277 


heating,  and  cemented  fast  with  a  little  fresh  collodion 
applied  to  the  line  of  union.  Novy  recommends  that 
a  thread  of  silk  be  wound  around  the 
point  of  union,  to  hold  the  collodion  in 
place  and  to  aid  in  handling  the  fin- 
ished sac.  It  now  appears  as  in  figure 
80,  b.  The  sac  is  next  filled  with  dis- 
tilled water  up  to  the  thread,  the  tube 
is  plugged  with  cotton,  and  the  whole 
placed  in  a  larger  test-tube  containing 
distilled  water,  the  cotton  plug  being 
packed  tightly  around  the  smaller 
tube,  so  that  the  collodion  sac  does 
not  reach  the  bottom  of  the  large 
tube,  but  hangs  suspended  in  the 
water  it  contains.  The  whole  is  now 
carefully  sterilized  by  steam. 

When  ready  for  use,  a  tube  of 
bouillon  is  inoculated  with  the  culture 
intended  to  be  placed  in  the  animal, 
the  water  in  the  capsule  is  pipetted 

out  and  replaced  by  the  inoculated  bouillon  carefully  intro- 
duced with  a  pipet,  the  constricted  portion  is  sealed  in  a 
flame,  and  the  capsule  picked  up  with  forceps  and'  intro- 
duced into  the  peritoneum  by  an  aseptic  operation. 

The  collodion  capsules  may  be  made  of  any  size.  Those 
for  rabbit  experiments  should  be  of  about  id  c.c.  capacity, 
those  for  guinea-pig  experiments  about  5  c.c.  By  coating 
large  glass  tubes  they  can  be  made  of  500  c.c.  capacity, 
the  large  bags  being  useful  for  chemic  dialysis. 


Fig.  80. — Prepara- 
tion of  collodion  sacs: 
a,  Test-tube  constric- 
ted and  cut;  b,  sac 
attached  to  the  tube. 


CHAPTER   XI. 
THE  DETERMINATION  OF  BACTERIA. 

THE  most  difficult  thing  in  bacteriology  is  the  determina- 
tion of  the  species  of  bacteria  that  come  under  observation. 

A  few  micro-organisms  are  characteristic  in  morphology 
and  in  their  chemic  and  other  products,  and  present  no  diffi- 
culty. Thus,  the  tubercle  bacillus  is  characteristic  in  its 
reaction  to  the  anilin  dyes,  and  can  usually  be  recognized  by 
this  peculiarity.  Some,  as  Bacillus  mycoides,  have  charac- 
teristic agar-agar  growths.  The  red  color  of  Bacillus  pro- 
digiosus  and  the  blue  of  Bacillus  janthinus  speak  almost 
positively  for  them.  The  potato  cultures  of  Bacillus  mesen- 
tericus  fuscus  and  vulgatus  are  usually  sufficient  to  enable 
us  to  recognize  them.  Unfortunately,  however,  there  are 
several  hundreds  of  described  species  that  lack  any  one  dis- 
tinct characteristic  that  may  be  used  for  differential  pur- 
poses, and  require  that  for  their  recognition  we  shall  well-nigh 
exhaust  the  bacteriologic  technic  in  order  to  identify  them. 

Tables  for  the  purpose  have  been  compiled  by  Eisenberg, 
Migula,  Lehman  and  Neumann,  Chester,  and  others,  and  are 
indispensable  to  the  worker.  The  most  useful  are  probably 
the  "Atlas  and  Grundriss  der  Bakteriologie  und  Lehrbuch 
der  speziellen  bakteriologischen  Diagnostik,"  by  Lehmann 
and  Neumann,*  and  the  "Manual  of  Determinative  Bacte- 
riology," by  F.  D.  Chester  (1901),  from  which,  through  the 
courtesy  of  the  author  and  publisher,  the  following  synopsis 
of  groups  is  taken.  Unfortunately,  in  tabulating  bacteria 
we  constantly  meet  species  described  so  insufficiently  as  to 
make  it  impossible  to  properly  classify  and  tabulate  them. 

The  only  way  to  determine  a  species  is  to  study  it  thor- 
oughly, step  by  step,  and  compare  it  with  the  description 
and  tables.  In  this  regard  the  differentiation  of  bacteria 
resembles  the  determination  of  the  higher  plants  with  the 
aid  of  a  botanic  key,  or  the  qualitative  analysis  for  the  de- 
tection of  unknown  chemic  compounds.  Such  a  key  for 
specific  bacterial  differentiation  is  really  indispensable,  even 
though  it  be  imperfect,  and  every  student  engaged  in  research 

*  J.  F.  Lehmann,  Miinchen,  1907. 
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SUGAR  BOUILLON  IN 
FERMENTATION-TUBES 

Dextrose  
Lactose  
Saccharose  

GMENT, 

developed  in  pres< 
"  in  
color  ,  ch 
soluble  in  , 

•TIMUM  TEMPERATURE  .. 

PRODUCTION  OF  ACIDS  < 
Carbohydrates  absent 

^ 

£ 

O 

Chester's  Synopsis  of  Groups  of  Bacteria     279 

work  should  have  one.  As  Chester  says,  "probably  nine- 
tenths  of  the  forms  of  bacteria  already  described  might  as 
well  be  forgotten  or  given  a  respectful  burial.  This  will  then 
leave  comparatively  few  well-defined  species  to  form  the 
nuclei  of  groups  in  one  or  another  of  which  we  shall  be  able 
to  place  all  new  and  sufficiently  described  forms."  "That 
typical  forms  or  species  of  bacteria  do  exist,  no  one  can  deny. 
These  typical  forms  furthermore  present  certain  definite 
morphologic,  biologic,  cultural,  and  perhaps  pathogenic 
characters  which  establish  the  types  independently  of  minor 
variations. 

"The  most  marked  of  these  types  we  select  to  become  the 
centers  of  groups,  around  which  are  gathered  all  related 
species  or  varieties."  "The  division  of  the  bacteria  into 
groups,  so  far  as  grouping  was  possible,  is  outlined  in  the  fol- 
lowing tables:" 

A  PROPOSED   SYNOPSIS  OF  GROUPS  OF   BACTERIA. 

BACTERIUM. 
I.  Without  endospores. 

A.  Aerobic  and  facultative  anaerobic, 
a.  Gelatin  not  liquefied. 

*  Decolorized  by  Gram's  method. 

t  Obligate  aerobic.     ACETIC  FERMENT  GROUP. 
ft  Aerobic  and  facultative  anaerobic. 
Gas  generated  in  glucose  bouillon. 

Gas    generated    in    lactose    bouillon.         BACT. 

AEROGENES  GROUP. 
Little  or  no  gas  generated  in  lactose  bouillon. 

FRIEDLANDER  GROUP. 
No  gas  generated  in  glucose  bouillon. 

Milk  coagulated.     FOWL  CHOLERA  GROUP. 
Milk  not  coagulated.     SWINE  PLAGUE  GROUP. 
**  Stained  by  Gram's  method. 

f  Gas   generated   in    glucose    bouillon.     LACTIC    FER- 
MENT GROUP. 
6.  Gelatin  liquefied. 

*  Colonies  on   gelatin   ameboid   or  proteus-like.     BACT. 

RADIATUM  GROUP. 

**  Colonies  on  gelatin  round,  not  ameboid.     BACT.  AM- 

BIGUUM  GROUP. 
II.  Produce  endospores. 

1.  No  growth  at  room  temperature,  or  below  22°-25°  C.     THER- 

MOPHILIC  GROUP. 

2.  Grow  at  room  temperatures. 

a.  Gelatin  liquefied.     ANTHRAX  GROUP. 

6.  Gelatin  not  liquefied.     BACT.  F^CALIS  GROUP. 

BACILLUS. 
I.  Without  endospores. 

A.  Aerobic  and  facultative  anaerobic. 


280  The  Determination  of  Bacteria 

0.  Gelatin  colonies  roundish,  not  distinctly  ameboid. 

*  Gelatin  not  liquefied. 

f  Decolorized  by  Gram's  method. 

Gas  generated  in  glucose  bouillon. 
Milk  coagulated.     COLON  GROUP. 
Milk  not  coagulated.     HOG  CHOLERA  GROUP. 
No  gas  generated  in  glucose  bouillon.     TYPHOID 

GROUP. 
tt  Stained     by     Gram's     method.     B.     MURIPESTIFER 

GROUP. 
**  Gelatin  liquefied. 

f  Gas  generated  in  glucose  bouillon.    B.  CLOACA  GROUP. 

ft  No   gas   generated   in   glucose   bouillon.     Include   a 

large  number  of  bacteria  not  sufficiently  described 

to  arrange  in  groups. 

b.  Gelatin  colonies  ameboid,  cochleate,  or  otherwise  irregular. 

*  Gelatin  liquefied.     PROTEUS  VULGARIS  GROUP. 
**  Gelatin  not  liquefied.     B.  ZOPFI  GROUP. 

fl.  Produce  endospores. 

A.  Aerobic  and  facultative  anaerobic. 

1.  Rods  not  swollen  at  sporulation. 

a.  Gelatin  liquefied. 

*  Liquefaction  of  the  gelatin  takes  place  slowly.     Fer- 
ment urea,  with  strong  production  of  ammonia. 
URO-BACILLUS  GROUP  OF  MIQUEL. 
**  Gelatin  liquefied  rather  quickly. 

t  Potato  cultures  rugose.     POTATO  BACILLUS  GROUP. 
tf  Potato  cultures  not  distinctly  rugose.     B.  SUBTILIS 

GROUP. 
b.  Gelatin  not  liquefied.     B.  SOLI  GROUP. 

2.  Rods  spindle-shaped  at  sporulation.     B.   LICHENIFORMIS 

GROUP. 

3.  Rods  clavate  at  sporulation.     B.  SUBLANATUS  GROUP. 

B.  Obligate  anaerobic. 

1.  Rods   not    swollen   at    sporulation.     MALIGNANT   EDEMA 

GROUP. 

2.  Rods  spindle-shaped  at  sporulation.    CLOSTRIDIUM  GROUP. 

3.  Rods  clavate-capitate  at  sporulation.     TETANUS  GROUP. 

PSEUDOMONAS  (Migula). 

I.  Cells  colorless,  without  a  red-colored  plasma  and  without  sulphur 

granules. 

A.  Grow  in  ordinary  culture  media. 
1.  Without  endospores. 

a.  Aerobic  and  facultative  anaerobic. 

*  Without  pigment. 

t  Gelatin  not  liquefied. 

Gas  generated  in  glucose  bouillon.     Ps.  MONADI- 

FORMIS  GROUP. 
No  gas  generated  in  glucose  bouillon.     Ps.  AM- 

BIGUA  GROUP. 
tt  Gelatin  liquefied. 

Gas  generated  in  glucose  bouillon.     Ps.   COADU- 

NATA  GROUP. 
No  gas  generated  in  glucose  bouillon.     Ps.  FAIR- 

MONTENSIS  GROUP. 

*  Produce  pigment  on  gelatin  or  agar. 
t  Pigment  yellowish. 


Chester's  Synopsis  of  Groups  of  Bacteria    281 

Gelatin  liquefied.     Ps.  OCHRACEA  GROUP. 
Gelatin  not  liquefied.     Ps.  TURCOSA  GROUP. 
ft  Pigment  blue-violet. 

Gelatin  liquefied.     Ps.  JANTHINA  GROUP. 
Gelatin  not  liquefied.    Ps.  BEROLINENSIS  GROUP. 
**  Produce  a  greenish-bluish  fluorescence  in  culture  media. 

f  Gelatin  liquefied.     Ps.  PYOCYANEA  GROUP. 
ft  Gelatin  not  liquefied.     Ps.  SYNCYANEA  GROUP. 
2.  With  endospores,  aerobic  and  facultative  anaerobic. 

a.  Non-chromogenic. 

*  Rods  not  swollen  at  sporulation.    Ps.  ROSEA  GROUP. 
**  Rods  swollen  at  one  end  at  sporulation.     Ps.  TROM- 

MELSCHLAGER    GROUP. 

b.  Produce  a  greenish-bluish  fluorescence  in  culture  media. 

*  Gelatin  liquefied.     Ps.  VIRIDESCENS  GROUP. 
**  Gelatin  not  liquefied.     Ps.  UNDULATA  GROUP. 

B.  Do  not  grow  in  nutrient  gelatin  or  other  organic  media.     Ni- 

TRIMONAS  GROUP. 

II.  Cell  plasma  with  a  reddish  tint,  also  with  sulphur  granules.  CHRO- 
MATIUM  GROUP. 

MICROSPIRA  (Migula). 
I.  Cultures  show  a  bluish-silvery  phosphorescence.    PHOSPHORESCENT 

GROUP. 
II.  Cultures  not  phosphorescent. 

A.  Gelatin  liquefied. 

1.  Cultures  show  the  nitro-indol  reaction. 

a.  Very     pathogenic     to     pigeons.      MSP.     METSCHNIKOVI 

GROUP. 

b.  Not  distinctly  pathogenic  to  pigeons.     CHOLERA  GROUP. 

2.  Nitro-indol  reaction  negative  or  very  weak,  at  least  after 

twenty-four  hours.     CHOLERA  NOSTRAS  GROUP. 

B.  Gelatin  not  liquefied  or  only  slightly  so.     MSP.  SAPROPHILA 

GROUP. 

MYCOBACTERIUM  (Lehmann- Neumann). 

I.  Stain  with  basic  anilin  dyes,  and  easily  decolorized  by  mineral 
acids  when  stained  with  carbol-fuchsin. 

A.  Grow  well  on  nutrient  gelatin.     Gelatin  liquefied  very  slowly 

or  merely  softened. 

1.  Stain  by  Gram's  method.     SWINE  ERYSIPELAS  GROUP. 

2.  Not  stained  by  Gram's  method.     GLANDERS  GROUP. 

B.  Little  or  no  growth  in  ordinary  nutrient  gelatin. 

1.  Grow  well  in  nutrient  bouillon  at  body  temperatures. 

a.  Stained  by  Gram's  method.     Rods  cuneate — clavate — 
irregularly  swollen.     DIPHTHERIA  GROUP. 

2.  No  growth  in  nutrient  bouillon  or  on  ordinary  culture  media. 

Rods  slender,  tubercle-like, 
a.  Stain  by  Gram's  method.     LEPROSY  GROUP. 
6.  Do  not  stain  by  Gram's  method.     INFLUENZA  GROUP. 

3.  No  growth  in  nutrient  bouillon  or  on  ordinary  culture 

media.     Rods  variable.     ROOT-TUBERCLE  GROUP. 
II.  Not  stained  with  aqueous  solutions  of  basic  anilin  dyes;  not  easily 
decolorized  by  acids.     TUBERCLE  GROUP. 

COCCACE^E. 

Cells  in  their  free  condition  globular,  becoming  slightly  elongated 
before  division.  Cell  division  in  one,  two,  or  three  directions 
of  space. 


282  The  Determination  of  Bacteria 

A.  Cells  without  flagella. 

1.  Division   in   only   one   direction   of   space.     Streptococcus 

(Billroth). 

2.  Division  in  two  directions  of  space.     Micrococcus  (Hallier). 

3.  Division  in  three  directions  of  space.     Sarcina  (Goodsir). 

B.  Cells  with  flagella. 

1.  Division  in  two  directions  of  space.     Planococcus  (Migula). 

2.  Division    in    three    directions    of    space.      Planosarcina 

(Migula). 


CHAPTER  XII. 

THE  BACTERIOLOGY  OF  THE  AIR, 

MICRO-ORGANISMS  are  almost  universally  suspended  in  the 
dust  of  the  air,  their  presence  being  a  constant  source  of  con- 
tamination in  our  bacteriologic  researches  and  occasionally 
a  menace  to  our  health. 

Such  aerial  organisms  are  neither  ubiquitous  nor  uni- 
formly disseminated,  but  are  much  more  numerous  where 
the  air  is  polluted  and  dusty  than  where  it  is  pure.  The 
purity  of  the  atmosphere  bears  a  distinct  relation  to  the 
purity  of  the  surfaces  over  which  its  currents  blow. 

The  micro-organisms  of  the  air  are  for  the  most  part  harm- 
less saprophytes  taken  up  and  carried  about  by  the  wind. 
They  are  almost  always  taken  up  from  dry  materials,  ex- 
periment having  shown  that  they  arise  from  the  surfaces  of 
liquids  with  much  difficulty.  Not  all  the  micro-organisms 
of  the  air  are  bacteria,  and  a  plate  of  sterile  gelatin  exposed 
to  the  air  for  a  brief  time  will  generally  grow  molds  and  yeasts 
as  well. 

In  some  cases  the  bacteria  are  pathogenic,  especially  where 
discharges  from  diseased  animals  have  been  allowed  to  collect 
and  dry.  On  this  account  the  atmosphere  of  hospital  wards 
and  of  rooms  in  which  infectious  diseases  are  being  treated  is 
more  apt  to  contain  them  than  the  air  of  the  street.  How- 
ever, because  of  the  expectoration  from  cases  of  tuberculosis, 
influenza,  and  pneumonia,  which  is  often  ejected  upon  the 
sidewalks  and  floors  of  public  places,  the  presence  of  occa- 
sional pathogenic  bacteria  is  far  from  uncommon  in  street- 
dust. 

Gunther  points  out  that  the  greater  number  of  the  bacteria 
which  occur  in  the  air  are  cocci,  sarcina  being  particularly 
abundant.  Most  of  them  are  chromogenic  and  do  not  liquefy 
gelatin.  It  is  unusual  to  find  more  than  two  or  three  varie- 
ties of  bacteria  at  a  time. 

To  determine  whether  bacteria  are  present  in  the  air  or 
not,  all  that  is  necessary  is  to  expose  a  film  of  sterile  gelatin 

283 


284  The  Bacteriology  of  the  Air 

on  a  plate  or  Petri  dish  to  the  air  for  a  while,  cover,  and 
observe  whether  or  not  bacteria  grow  upon  it. 

To  make  a  quantitative  estimation  is,  however,  more  diffi- 
cult. Several  methods  have  been  suggested,  of  which  the 
most  important  may  be  briefly  mentioned : 

Hesse's  method  is  simple  and  good.  It  consists  in  mak- 
ing a  measured  quantity  of  the  air  to  be  examined  pass 
through  a  horizontal  sterile  glass  tube  about  70  cm.  long  and 
3.5  cm.  wide  (Fig.  81),  the  interior  of  which  is  coated  with  a 
film  of  gelatin  in  the  same  manner  as  an  Ksmarch  tube.  The 


Fig.  81. — Hesse's  apparatus  for  collecting  bacteria  from  the  air. 

tube  is  closed  at  both  ends  with  sterile  corks  carrying  small 
glass  tubes  plugged  with  cotton.  When  ready  for  use  the 
tube  at  one  end  is  attached  to  a  hand-pump,  the  cotton  re- 
moved from  the  other  end,  and  the  air  slowly  passed  through, 
the  bacteria  having  time  to  sediment  upon  the  gelatin  as 
they  pass.  When  the  required  amount  has  passed,  the 
tubes  are  again  plugged,  the  apparatus  stood  away  for  a 
time,  and  subsequently,  when  they  have  grown,  the  colonies 
are  counted.  The  number  of  colonies  in  the  tube  will  repre- 
sent pretty  accurately  the  number  of  bacteria  in  volume  of 
air  that  passed  through  the  tube. 


Petri's  Method 


285 


In  such  a  tube,  if  the  air  pass  through  with  proper  slowness, 
the  colonies  will  be  much  more  numerous  near  the  point  of 
entrance  than  near  that  of  exit.  The  first  to  fall  will  prob- 
ably be  those  of  heaviest  specific  gravity — i.  e.,  the  molds. 

Petri's  Method. — A  more  exact  method  is  that  of  Petri, 
who  uses  small  filters  of  sand  held  in  place  in  a  wide  glass 
tube  by  small  wire  nets  (Fig. 
82).     The  sand  used  is  made 
to  pass  through  a  sieve  whose 
openings  are  of  known  size, 
is  heated  to   incandescence, 
then  arranged  in  the  tube  so 
that  two  of  the  little  filters, 
held  in  place  by  their  wire- 


Fig.  82.— Petri's  sand  filter  for 
air-examination. 


Fig.    83. — Sedgwick's    expanded 
tube  for  air-examination. 


gauze  coverings,  are  superimposed.  One  or  both  ends  of  the 
tube  are  closed  with  corks  having  a  narrow  glass  tube.  The 
apparatus  is  sterilized  by  hot  air,  and  is  then  ready  for  use. 
The  method  of  employment  is  very  simple.  By  means  of 
a  hand-pump  100  liters  of  air  are  made  to  pass  through  the 
filter  in  from  ten  to  twenty  minutes,  the  contained  micro- 
organisms being  caught  and  retained  by  the  sand.  The  sand 


286  The  Bacteriology  of  the  Air 

from  the  upper  filter  is  then  carefully  mixed  with  sterile 
melted  gelatin  and  poured  into  sterile  Petri  dishes,  where 
the  colonies  develop  and  can  be  counted.  Petri  points  out 
in  relation  to  his  method  that  the  filter  catches  a  relatively 
greater  number  of  bacteria  in  proportion  to  molds  than  the 
Hesse  apparatus,  which  depends  upon  sedimentation.  Stern- 
berg  points  out  that  the  chief  objection  to  the  method  is  the 
presence  of  the  sand,  which  interferes  with  the  recognition 
and  counting  of  the  colonies  in  the  gelatin. 

Sedgwick's  Method. — Sedgwick  and  Miquel  have  recom- 
mended the  use  of  a  soluble  material — granulated  or  pulver- 
ized sugar — instead  of  the  sand.  The  apparatus  used  for 
the  sugar  experiments  differs  a  little  from  the  original  of 
Petri,  though  the  principle  is  the  same,  and  can  be  modified 
to  suit  the  experimenter. 

A  particularly  useful  form  of  apparatus  (Fig.  83),  sug- 
gested by  Sedgwick  and  Tucker,  has  an  expansion  above  the 
filter,  so  that  as  soon  as  the  sugar  is  dissolved  in  the  melted 
gelatin  it  can  be  rolled  out  into  a  film  like  that  of  an  Esmarch 
tube.  This  cylindric  expansion  is  divided  into  squares 
which  make  the  counting  of  the  colonies  very  easy. 

Roughly,  the  number  of  germs  in  the  atmosphere  may  be 
estimated  at  from  100  to  1000  per  cubic  meter. 

The  bacteriologic  examination  of  air  is  of  very  little  im- 
portance because  of  the  numerous  errors  that  must  be  met. 
Thus,  when  the  air  of  a  room  is  quiescent  it  may  contain 
very  few  bacteria ;  let  some  one  walk  across  the  floor  so  that 
dust  rises,  and  the  number  of  bacteria  becomes  considerably 
increased;  if  the  room  be  swept,  the  increase  is  enormous. 
From  these  and  similar  contingencies  it  becomes  very  diffi- 
cult to  know  just  when  and  how  the  air  is  to  be  examined, 
and  the  value  of  the  results  is  correspondingly  lessened. 

The  most  sensible  studies  of  the  air  aim  rather  at  the  dis- 
covery of  some  definite  organism  or  organisms  than  at  the 
determination  of  the  total  number  per  cubic  meter. 


CHAPTER   XIII. 

BACTERIOLOGY  OF  WATER. 

UNLESS  water  has  been  specially  sterilized,  received  and 
kept  in  sterile  vessels,  it  always  contains  some  bacteria,  the 
number  usually  bearing  a  distinct  relationship  to  the  quan- 
tity of  organic  matter  present. 

The  majority  of  the  water  bacteria  are  bacilli,  and  are  as  a 
rule  non-pathogenic.  Wright,*  in  his  examination  of  the 
bacteria  of  the  water  from  the  Schuylkill  River,  found  two 
species  of  micrococci,  two  species  of  cladothrices,  and  forty- 
six  species  and  two  varieties  of  bacilli.  Pathogenic  bacteria, 
such  as  the  spirillum  of  Asiatic  cholera  and  the  bacillus  of 


Fig.  84. — Wolfhiigel's  apparatus  for  counting  colonies  of  bacteria  upon 

plates. 

typhoid  fever,  may  occur  in  polluted  water,  but  their  occur- 
rence is  exceptional. 

The  method  of  determining  the  number  of  bacteria  in 
water  is  very  simple,  and  can  be  accomplished  with  very 
little  apparatus.  The  method  depends  upon  the  equal  dis- 
tribution of  a  measured  quantity  of  the  water  to  be  exam- 
ined in  some  sterile  liquefied  medium,  whose  subsequent 
solidification  in  a  thin  layer  permits  the  colonies  to  be 
counted. 

The  method  originated  with  Koch,  and  may  be  performed 
with  plates,  Petri  dishes,  or  Esmarch  rolls.  It  is  always  best 

*  "Memoirs  of  the  National  Academy  of  Sciences,"  vol.  vn,  Third 
Memoir. 

287 


288 


Bacteriology  of  Water 


to  make  a  number  of  cultures  with  different  quantities  of  the 
water,  using,  for  example,  o.oi,  o.i,  0.5,  and  i.o  c.c.,  re- 
spectively, to  a  tube  of  liquefied  gelatin,  agar-agar,  or  gly- 
cerin agar-agar. 

The  details  of  the  method  depend  upon  the  quality  of  the 
water  to  be  examined.  If  the  number  of  bacteria  per  cubic 
centimeter  be  small,  large  quantities  may  be  used ;  but  if 
there  be  millions  of  bacteria  in  every  cubic  centimeter,  it 
may  be  necessary  to  dilute  the  water  to  be  examined  in  the 
proportion  of  i :  10  or  i :  100  with  sterile  water,  mixing  well, 
and  making  the  plate  cultures  from  the  dilutions. 

It  is  best  to  count  all  the  colonies  developed  upon  the 

culture,  if  possible;  but  when 
hundreds  of  thousands  are  scat- 
tered over  it,  an  estimate  made 
by  counting  and  averaging  the 
number  in  each  of  the  small 
squares  of  some  counting  appa- 
ratus, such  as  those  devised  by 
Wolfhiigel  (Fig.  84),  Esmarch 
(Fig.  85),  or  Frost  (Fig.  86). 
In  counting  the  colonies  a  lens 
is  indispensable. 

In  some  cases,  as  in  the  study 
of  sewage,  badly  contaminated 
waters,  inflammatory  exudates, 
and  in  the  preparation  of  bac- 
terial vaccines,  it  is  expedient 
to  directly  enumerate  the  bac- 
teria without  resorting  to  the  cultivation  method,  where  all 
of  the  organisms  may  not  grow. 

Excellent  methods  for  achieving  this  have  been  devised. 
That  of  Winslow  and  Willcomb*  being  as  follows: 

"The  cover-slips  should  be  boiled  in  a  10  per  cent,  solution 
of  potassium  bichromate  in  50  per  cent,  sulphuric  acid  and 
allowed  to  lie  in  this  cleansing  mixture.  Just  before  using 
they  may  be  rinsed  in  50  per  cent,  alcohol  and  dried  on  a 
silk  cloth,  not  in  the  flame.  One- twentieth  of  a  cubic  centi- 
meter of  water  placed  on  such  a  cover-slip  spreads  evenly 
and  should  be  allowed  to  dry  in  the  air  without  sudden 
heating.  After  drying  it  is  fixed  by  passing  through  the 
flame,  covered  with  Ziehl-Neelson's  carbol-fuchsin,  warmed 
*  "Jour,  of  Infectious  Diseases,"  Supplement  No.  i,  May,  1905,  p.  273. 


Fig.  85. — Esm arch's  instru- 
ment for  counting  colonies  of 
bacteria  in  Esmarch  tubes. 


Determination  of  Bacteria  in  Water          289 

until  steam  just  rises,  washed,  dried,  and  mounted.  For 
counting  the  bacteria  we  use  a  Sedgwick-Rafter  eye-piece 
micrometer,  made  for  the  study  of  the  larger  micro-organisms 
in  drinking  water."  Very  uniform  results  have  followed. 

The  method  of  Wright*  was  devised  for  the  computation 
of  bacteria  in  suspensions  used  in  making  tests  of  the  opsonic 
power  of  the  blood  and  for  use  as  bacterio-vaccines.  The 
bacteria  containing  fluid  is  diluted  as  may  be  necessary 
with  an  equal  volume  or  i :  2,  1:5,  i :  10,  with  a  fluid  of  which 
one  volume  is  normal  human  blood,  then  spread  upon  a 
slide,  fixed  and  stained  by  Irishman's  or  some  other  appro- 
priate method,  placed  upon  the  stage  of  the  microscope, 
and  the  number  of  blood-corpuscles  and  bacteria  counted 
in  each  of  a  number  of  fields.  To  facilitate  the  counting  a 
cross  is  ruled  with  a  writing  diamond,  upon  a  circular  cover- 
glass,  which  is  then  dropped  into  the  eye-piece.  If  there 
are,  for  example,  ten  bacteria  to  each  corpuscle  in  a  1:5 
dilution,  the  calculation  is  based  upon  the  number  of  cor- 
puscles. There  being  5,000,000  red  corpuscles  in  i  c.mm.  of 
normal  blood,  there  should  be  50,000,000  bacteria  in  i  c.mm. 
of  the  suspension,  but  as  it  has  been  diluted  1:5,  there  are 
only  -J-  of  this,  or  10,000,000. 

The  majority  of  the  water  bacteria  rapidly  liquefy  gelatin, 
on  which  account  it  is  better  to  employ  both  gelatin  and 
agar-agar  in  making  the  cultures. 

In  ordinary  city  hydrant-water  the  bacteria  number  from 
2  to  50  per  cubic  centimeter;  in  good  pump- water,  100  to  500; 
in  filtered  water  from  rivers,  according  to  Giinther,  50  to  200; 
in  unfiltered  river- water,  6000  to  20,000.  According  to  the 
pollution  of  the  water  the  number  may  reach  as  many  as 
50,000,000. 

The  waters  of  wells  and  springs  are  dependent  for  their 
purity  upon  the  character  of  the  earth  or  rock  through  which 
they  filter,  and  the  waters  of  deep  wells  are  much  more  pure 
than  those  of  shallow  wells,  unless  contamination  take  place 
from  the  surface  of  the  ground. 

Ice  always  contains  bacteria  if  the  water  contained  them 
before  it  was  frozen.  In  Hudson  River  ice  Prudden  found 
an  average  of  398  colonies  in  a  cubic  centimeter. 

A  sample  of  water  when  collected  for  examination  should 
be  placed  in  a  clean  sterile  bottle  or  in  a  hermetically  sealed 
pre-sterilized  glass  bulb,  and  must  be  examined  as  soon  as 
possible,  as  the  bacteria  multiply  rapidly  in  water  which  is 

*  "Lancet,"  July  5,  1902. 
19 


290  Bacteriology  of  Water 

allowed  to  stand  for  a  short  time.  If  the  water  to  be  exam- 
ined must  be  transported  any  considerable  distance  before 
the  manipulations  are  performed,  it  should  be  packed  in  ice. 
The  greatest  care  must  always  be  exercised  that  the  unnat- 
ural conditions  arising  from  the  bottling  of  the  water,  the 
changes  of  temperature,  and  the  altered  relationship  to  light 
and  the  atmosphere,  do  not  modify  the  number  of  contained 
bacteria. 


100 


Fig.  86. — Frost's  plate  counter,  for  counting  colonies  of  bacteria  on 
Petri  dish  or  plate  cultures.  The  cross-lines  divide  the  figure  into 
square  centimeters.  The  numbers  at  the  top  of  the  figure  indicate  the 
area  in  centimeters  of  the  various  discs.  The  area  of  each  sector  (a  and 
b)  is  one-tenth  of  the  whole  area. 

The  detection  of  such  important  bacteria  as  the  colon  and 
typhoid  bacilli,  and  the  cholera  spirillum,  will  be  considered 
in  the  chapters  treating  of  those  respective  organisms. 

Unfortunately,  the  bacteriologic  examination  of  waters 
does  not  throw  satisfactory  light  upon  their  exact  hygienic 
usefulness.  Of  course,  if  cholera  or  typhoid  fever  bacteria 
are  present,  the  water  is  dangerous,  but  the  quality  of  the 
water  cannot  always  be  gauged  by  the  number  of  bacteria  it 
contains. 


Determination  of  Bacteria  in  Water         291 

Drinking-water,  especially  that  furnished  to  large  cities, 
is  not  infrequently  contaminated  with  sewage,  and  contains 
intestinal  bacteria — Bacillus  coli  communis.  For  the  ready 
determination  of  this  organism,  which  is  an  important  indi- 
cation that  the  water  is  polluted,  Smith  *  has  made  use  of 
the  fermentation-tube  in  addition  to  the  plate.  His  method 
is  to  add  to  each  of  several  fermentation-tubes  containing 
i  per  cent,  dextrose-bouillon  a  certain  quantity  of  water. 
The  evolution  of  50-60  per  cent,  of  gas  by  the  third  day  is  a 
strong  indication  that  the  colon  bacillus  is  present.  Plates 
may  be  used  to  confirm  the  presence  of  the  bacillus,  but  are 
hardly  necessary,  as  there  is  scarcely  another  bacterium  met 
with  in  water  that  is  capable  of  producing  so  much  gas. 

It  was  at  one  time  thought  that  the  occurrence  of  the  colon 
bacillus  in  water  was  sufficient  to  condemn  its  potability,  but 
the  evidence  accumulated  in  recent  years,  showing  that  this 
organism  may  reach  streams  from  manured  soil,  may  enter 
it  with  the  dejecta  of  domestic  animals,  wild  animals,  birds, 
and  perhaps  even  of  fishes,  makes  it  doubtful  whether  any- 
thing but  an  exceptionally  large  number  of  the  organisms 
should  be  looked  upon  as  indicative  of  sewage  pollution  and 
proof  that  the  water  is  not  potable. 

In  determining  the  species  of  bacteria  found  in  the  water 
reference  must  be  made  to  the  numerous  monographs  upon 
the  subject  and  to  special  tables.  An  excellent  table  of  this 
kind,  arranged  by  Fuller,  f  is  given  on  pages  292,  293. 

Filtration  with  sand,  etc.,  diminishes  the  number  of  bac- 
teria for  a  time,  but,  as  the  organisms  multiply  in  the  filter, 
the  benefit  is  not  permanent  and  the  filters  must  frequently 
be  subjected  to  bacteriologic  tests  and  the  sand  washed, 
spread  out  to  dry  and  the  filters  renewed.  Porcelain  filters 
seem  to  be  the  only  positive  safeguard,  and  even  these,  the 
best  of  which  seems  to  be  the  Pasteur-Chamberland,  allow 
the  bacteria  to  pass  through  if  used  too  long  without  proper 
attention. 

For  those  whose  special  line  of  work  is  the  bacteriology  of 
water,  the  report  of  the  Committee  on  Standard  Methods 
of  Water  Analysis  to  the  Laboratory  Section  of  the  Ameri- 
can Public  Health  Association,  published  in  Supplement 
No.  i  of  the  "Journal  of  Infectious  Diseases,"  May,  1905, 
will  prove  indispensable. 

*  "  Amer.  Jour.  Med.  Sci.,"  1895,  no,  p.  301. 

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CHAPTER   XIV. 
BACTERIOLOGY  OF  THE  SOIL. 

THE  upper  layers  of  the  soil  contain  bacteria  in  proportion 
to  their  richness  in  organic  matter.  Near  the  habitations  of 
men,  where  the  soil  is  cultivated,  the  excrement  of  animals, 
largely  made  up  of  bacteria,  is  spread  upon  it  to  increase  its 
fertility,  this  treatment  not  only  adding  new  bacteria  to 
those  already  present,  but  also  enabling  those  present  to 
grow  much  more  luxuriantly  because  of  the  increased  nour- 
ishment they  receive. 

Where,  as  in  Japan,  human  excrement  is  used  to  fertilize 
the  soil,  or  as  in  India,  it  is  carelessly  deposited  upon  the 
ground,  bacteria  of  cholera,  dysentery,  and  typhoid  fever 
are  apt  to  become  disseminated  by  fresh  vegetables,  or 
through  water  into  which  the  soil  drains.  In  such  localities 
fresh  vegetables  should  not  be  eaten,  and  water  for  drinking 
should  be  boiled. 

The  researches  of  Fliigge,  C.  Frankel,  and  others  show  that 
the  bacteria  of  the  soil  do  not  penetrate  deeply,  but  gradu- 
ally decrease  in  number  until  the  depth  of  a  meter  is  reached, 
then  rapidly  diminish  until  at  a  meter  and  a  quarter  they 
rather  abruptly  disappear. 

The  bacteria  of  soil  are,  for  the  most  part,  harmless  sapro- 
phytes, though  a  few  highly  pathogenic  organisms,  such  as 
the  bacilli  of  tetanus  and  malignant  edema,  occur.  Many 
of  them  are  anaerobic,  and  it  is  interesting  to  speculate  upon 
their  biology.  Whether  they  develop  and  multiply  in  the 
soil  in  intimate  association  with  strongly  aerobic  organisms 
by  which  the  free  oxygen  is  absorbed,  or  whether  they  re- 
main latent  in  the  soil  and  develop  only  in  the  intestines  of 
animals,  is  not  known. 

The  estimation  of  the  number  of  bacteria  in  the  soil  seems 
to  be  devoid  of  any  practical  importance.  C.  Frankel  has, 
however,  originated  an  accurate  method  of  determining  it. 
By  means  of  a  special  boring  apparatus  (Fig.  87)  earth  can 
be  secured  from  any  depth  without  digging  and  without 
danger  of  mixing  with  that  of  the  superficial  strata.  A 

294 


Bacteria  in  Soil 


295 


measured  quantity  of  the  secured  soil  is  thoroughly  mixed 
with  liquefied  sterile  gelatin  and  poured  into  a  Petri  dish 
or  solidified  upon  the  walls  of  an  Esmarch  tube.  The 
colonies  are  counted  with  the  aid  of  a  lens.  Fliigge  found 
in  virgin  earth  about  100,000  colonies  in  a  cubic  centimeter. 

Samples  of  earth,  like  samples  of  water,  should  be  exam- 
ined as  soon  as  possible  after  being  secured,  for,  as  Gunther 
points  out,  the  number  of  bacteria  changes  because  of  the 
unusual  dryness,  warmth,  exposure  to  oxygen,  etc. 

The  most  important  bacteria  of  the 
soil  are  those  of  tetanus  and  malignant 
edema,  in  addition  to  which,  however, 
there  are  a  great  variety  of  organisms 
pathogenic  for  rabbits,  guinea-pigs,  and 
mice. 

In  the  "Bacteriological  Examination 
of  the  Soil  of  Philadelphia,"  Ravenel  * 
came  to  the  conclusion  that — 

1.  Made  soils,  as  commonly  found,  are 
rich  in  organic  matter  and   excessively 
damp  through  poor  drainage. 

2.  They  furnish  conditions  more  suited 
to  the  multiplication  of  bacteria  than  do 
virgin  soils,  unless  the  latter  are  contami- 
nated by  sewage  or  offal. 

3.  Made  soils  contain  large  numbers 
of  bacteria  per  gram  of  many  different 
species,  the  deeper  layers  being  as  rich 
in  the  number  and  variety  of  organisms 
as  the  upper   ones.     After   some   years 
the  number  in  the  deeper  layers  probably 
becomes  proportionally  less.     Made  soils 
are  more  likely  than  others  to  contain 
pathogenic  bacteria. 

In  seventy-one  cultures  that  were  iso- 
lated and  carefully  studied  by  Ravenel, 
there  were  two  cocci,  one  sarcina,  and 
five  cladothrices ;  all  the  others  were 
bacilli. 


Fig.  87.— Tip  of 
Frankel' s  instru- 
ment for  obtaining 
earth  from  various 
depths  for  bacterio- 
logic  study.  B 
shows  the  instru- 
ment with  its  cav- 
ity closed,  as  it 
appears  during  bor- 
ing; A,  open,  as  it 
appears  when  twis- 
ted in  the  other  di- 
rection to  collect 
the  earth. 


*  "Memoirs  of  the  National  Academy  of  Sciences,"  First  Memoir, 
1896. 


CHAPTER   XV. 

THE  BACTERIOLOGY  OF  FOODS. 

THE  relation  of  bacteria  to  foods  is  an  important  one  and 
should  be  as  thoroughly  understood  as  possible  by  both  the 
profession  and  the  laity.  The  relationship  may  be  expressed 
thus: 

I.  Foods  serve  as  vehicles  by  which  infectious  agents  are 
conveyed  to  the  body. 

II.  Foods  are  chemically  changed  and  made  unfit  for  use 
by  the  bacteria. 

I.  Foods  as  Fomites. — In  animal  food  the  first  source  of 
infection  is  the  animal  itself,  danger  of  infection  always 
accompanying  the  employment  of  foods  derived  from  dis- 
eased animals.  Thus,  milk  apparently  normal  in  appear- 
ance has  been  found  to  contain  dangerous  pathogenic  bac- 
teria. The  tubercle  bacillus  is  one  of  the  most  important  of 
these,  and  at  the  present  time  the  consensus  of  opinion  in- 
clines toward  the  view  that  the  great  prevalence  of  tubercu- 
losis among  human  beings  depends  partly  upon  the  inges- 
tion  of  tubercle  bacilli  in  milk.  It  does  not  appear  necessary 
that  the  udder  of  the  cow  be  diseased  in  order  that  the  or- 
ganisms enter  the  milk,  as  they  seem  to  have  been  found  in 
milks  derived  from  cows  whose  udders  were  entirely  free 
from  demonstrable  tuberculosis.  It  is,  therefore,  impera- 
tive to  retain  only  healthy  cows  in  the  dairy,  and  careful 
legislation  should  provide  for  the  detection  and  destruction 
of  all  tuberculous  animals.  The  detection  of  tubercle  bacilli 
in  milk  can  only  be  certainly  accomplished  by  the  injection 
of  a  few  cubic  centimeters  of  the  fluid  into  guinea-pigs  and 
noting  the  results. 

In  addition  to  the  tubercle  bacillus,  pyogenic  streptococci 
have  been  observed  in  enormous  quantities  and  almost  pure 
culture  in  milk  drawn  from  cows  suffering  from  mastitis, 
Stokes  *  has  observed  a  remarkable  case  of  this  kind  in  which 
the  milk  contained  so  much  pus  that  it  floated  upon  the  top 

*  "Maryland  Medical  Journal,"  Jan.  9,  1897. 
296 


Foods  as  Fomites  297 

like  cream.     Such  seriously  infected  milk  could  not  be  used 
with  safety  to  the  consumer. 

In  market  milk  one  occasionally  finds  pathogenic  organ- 
isms, such  as  the  diphtheria  bacillus,  typhoid  bacillus,  strep- 
tococcus, etc.,  derived  from  human  sources.  Such  polluted 
milks  have  been  known  to  spread  epidemics  of  the  respective 
diseases  whose  micro-organisms  are  present.  Bacteria  may 
enter  milk  from  careless  handling,  from  water  used  to  wash 
the  cans  or  to  dilute  the  milk,  or  from  dust ;  and  as  milk  is 
an  excellent  medium  for  the  growth  of  bacteria,  it  should 
always  be  treated  with  the  greatest  care  to  prevent  such  con- 
tamination, as  saprophytic  bacteria  produce  chemical 
changes  in  the  milk,  such  as  acidity  and  coagulation,  which 
destroy  its  usefulness  or  render  it  dangerous  as  food  for  in- 
fants and  invalids.  Where  the  necessary  precautions  are 
not  or  cannot  be  taken,  Pasteurization  of  the  milk  as  soon 
after  its  reception  as  possible  may  act  as  a  safeguard. 

The  student  interested  in  the  sanitary  relations  of  milk 
cannot  do  better  than  refer  to  Bulletin  No.  35  of  the  Hy- 
gienic Laboratory,  Washington,  D.  C.,  1907,  "Upon  the 
Origin  and  Prevalence  of  Typhoid  Fever  in  the  District  of 
Columbia,"  and  to  Bulletin  No.  41  of  the  same  laboratory, 
upon  "Milk  and  its  Relation  to  the  Public  Health"  (1908); 
also  to  the  "  Bacteriology  of  Milk,"  by  Swithinbank  and 
Neuman,  New  York,  E.  P.  Button  &  Co.,  1903. 

Meat  from  tuberculous  animals  might  cause  disease  if 
eaten  raw  or  but  partially  cooked.  As  cooking  suffices  to 
kill  the  organisms,  the  danger  under  ordinary  conditions  is 
not  great.  Moreover,  tuberculosis  rarely  affects  the  muscles, 
the  parts  usually  eaten. 

Butter  made  from  cream  derived  from  tuberculous  milk 
may  also  contain  tubercle  bacilli,  as  has  been  shown  by  the 
researches  of  Rabinowitsch.* 

Foods  may  become  polluted  with  bacteria  in  a  variety  of 
ways  that  will  suggest  themselves  to  the  reader.  The  com- 
mon source  is  dust,  which  is  more  or  less  rich  in  bacteria 
according  to  the  soil  from  which  it  arises.  The  readiness 
with  which  raw  foods,  such  as  meats,  milk,  etc.,  can  be  thus 
contaminated  in  the  barnyard,  dairy,  slaughter-house,  and 
shop,  teaches  but  one  lesson — that  the  greatest  cleanliness 
should  prevail  for  the  sake  of  the  dealer,  whose  goods  may  be 
*"  Deutsche  med.  Wochenschrift,"  1900,  No.  26;  abstract  in  the 
"Centralbl.  f.  Bakt.,"  etc.,  xxix,  1901,  p.  309. 


298  The  Bacteriology  of  Foods 

spoiled  by  carelessness,  and  the  consumer,  who  may  be  in- 
jured by  the  food. 

Any  food  may  carry  infectious  organisms  upon  its  surface, 
such  organisms  being  derived  from  the  hands  of  the  dealer, 
from  dust,  from  water,  as  when  green  vegetables  are  sprinkled 
with  impure  water  to  keep  them  fresh,  or  from  other  sources. 
The  cleanliness  of  the  merchant  and  the  protection  from 
contamination  that  he  bestows  upon  his  goods  should  be 
taken  into  consideration  by  his  customers. 

Shell-fish,  especially  oysters,  seem  to  be  common  carriers 
of  infection,  especially  of  typhoid  fever.  The  oysters  seem 
to  be  contaminated  with  infected  sewage  carried  to  their 
beds.  It  is  not  yet  satisfactorily  determined  whether 
typhoid  bacilli  multiply  in  the  juices  in  the  shells  of  the 
oysters  or  not,  but  a  number  of  epidemics  of  typhoid  fever 
have  been  very  conclusively  traced  to  the  consumption  of 
certain  oysters  at  a  definite  time  and  place.  As  cooking  the 
oysters  will  kill  the  contained  bacilli,  the  prophylaxis  of 
disease  in  this  case  is  very  simple. 

II.  Food  Poisons. — A  new  and  useful  nomenclature, 
suggested  by  Vaughan  and  Novy,*  contains  the  following 
terms : 

Bromatotoxismus — food-poisoning; 

Galactotoxismus — milk-poisoning; 

Tyrotoxismus — cheese-poisoning ; 

Kreotoxismus — meat-poisoning ; 

Ichthyotoxismus — fish-poisoning ; 

Mytilotoxismus — mussel-poisoning; 

Sitotoxismus — cereal-poisoning. 

The  most  important  chemic  alterations  effected  by  bac- 
teria occur  in  milk  and  meat. 

i.  Milk-poisoning  (Galactotoxismus). — Milk,  even  when 
freshly  drawn  from  the  cow,  always  contains  some  bacteria, 
whose  numbers  gradually  diminish  for  a  few  hours,  then  rap- 
idly increase  until  almost  beyond  belief.  These  organisms 
are  for  the  most  part  harmless  to  the  consumer,  but  ulti- 
mately ruin  the  milk.  Although  much  attention  has  been 
paid  to  the  subject,  bacteriologists  are  not  agreed  whether 
the  number  of  bacteria  contained  in  milk  is  a  satisfactory 
guide  as  to  its  harmfulness. 

The  poisonous  change  in  milk,  cream,  ice-cream,  etc., 
has  been  shown  by  Vaughan  to  depend  in  part  upon  the 
*"  Cellular  Toxins,"  Phila.,  1902. 


Food  Poisons  299 

presence  of  a  ptomain  known  as  tyrotoxicon,  formed  by 
the  growth  of  bacteria  in  the  milk,  but  whether  of  any 
particular  bacterium  is  not  known.  The  milk  may  become 
poisonous  during  any  time  of  the  year,  but  chiefly  in  the 
summer,  when,  because  of  the  higher  temperature,  bacteria 
develop  most  rapidly.  The  change  takes  place  in  stale  milk, 
and  it  is  supposed  that  many  cases  of  what  was  formerly 
looked  upon  as  "summer  complaint"  in  infants  were  really 
poisoning  by  this  toxic  ptomain. 

Ice-cream  poisoning  depends  upon  the  growth  of  the  bac- 
teria in  the  milk  before  it  is  frozen.  In  some  cases  the  error 
made  has  been  to  prepare  the  cream  for  freezing  and  then 
keep  or  transport  it,  the  freezing  operation  being  delayed 
until  the  development  of  the  bacteria  has  led  to  the  poison- 
ous condition. 

Cheese-poisoning  (Tyroloxismus)  is  also  thought  to  depend 
upon  tyrotoxicon  at  times,  though  it  has  been  shown  that 
other  cheese  poisons  exist.  It  is  more  or  less  a  question 
whether  cases  of  milk-  and  cheese-poisoning  do  not  depend 
upon  the  toxic  products  of  the  colon  bacillus  growing  in  the 
foods. 

2.  Meat-poisoning   (Kreotoxismus). — Botulism  or  meat- 
poisoning  depends  upon   the  growth  of  certain   bacteria, 
Bacillus  botulinus  of  van  Ermengem,*  in  the  meat.     The 
symptoms  following  infection  by  the  organism  sometimes 
closely  resemble  those  of  typhoid  fever,  and  are  characterized 
by  acute  gastro-intestinal  irritation,  nervous  disturbances, 
and,  in  case  of  death,  by  fatty  degenerations  in  the  organs 
and  minute  interstitial  hemorrhages. 

3.  Fish-poisoning    (Ichthyotoxismus)    sometimes   follows 
the  consumption  of  canned  and  presumably  spoiled  fish, 
sometimes   the   consumption  %  of  diseased   fish.     It  is  not 
known  whether  it  depends  upon  ptomains  or  upon  toxico- 
genic   germs,    though   probably   the   latter,    as   Silber  has 
isolated  a  Bacillus  piscicidus  that  is  highly  toxicogenic. 

4.  Mussel-poisoning    (Mytilotoxismus)    depends    partly 
upon  irritating  and  nervous  poisons  in  the  mussel  substance, 
in  part  upon  toxicogenic  germs  that  they  harbor. 

5.  Canned  Goods. — Improperly  preserved  canned  goods 
not  infrequently  spoil  because  of  the  growth  of  bacteria,  but 
the  occurrence  of    gas-formation,   acidity,  insipidity,   etc., 
causes  rejection  of  the  product,  and  but  few  cases  of  poison- 
ing from  canned  goods  can  be  authenticated. 

*  "Zeitschrift  fur  Hygiene,"  Bd.  xxvi,  Heft  1. 


CHAPTER   XVI. 

THE  DETERMINATION  OF  THE  THERMAL 
DEATH-POINT  OF  BACTERIA. 

SEVERAL  methods  may  be  employed  for  this  purpose. 
Roughly,  it  may  be  done  by  keeping  a  bouillon  culture  of 
the  micro-organism  to  be  investigated  in  a  water-bath  whose 
temperature  is  gradually  increased,  transplantations  being 
made  from  time  to  time  until  the  temperature  fatal  to 
the  bacteria  is  reached. 

It  is  economy  to  make  the  transplantations  less  frequently 
at  first  than  later  in  the  experiment,  when  the  ascending 
temperature  approaches  a  height  dangerous  to  life.  In  or- 
dinary determinations  it  is  well  to  make  a  transfer  at  40°  C., 
another  at  45°,  another  at  50°,  still  another  at  55°,  and  then, 
beginning  at  60°,  make  one  for  every  additional  degree.  The 
day  following  the  experiment  it  will  be  observed  that  all  the 
cultures  grow  except  those  heated  beyond  a  certain  point,  say 
62°  C.,  when  it  can  properly  be  concluded  that  62°  C.  is  the 
thermal  death-point.  If  all  the  transplantations  grow,  of 
course  the  maximum  temperature  was  not  reached,  and  the 
experiment  must  be  repeated  and  the  bacteria  exposed  to 
still  higher  temperatures. 

When  more  accurate  information  is  desired,  and  one 
wishes  to  know  how  long  the  micro-organism  can  endure 
some  such  temperature  as  60°  C.  without  losing  its  vitality, 
a  dozen  or  more  bouillon-tubes  may  be  inoculated  with  the 
organism  to  be  studied,  and  stood  in  a  water-bath  kept  at 
the  temperature  to  be  investigated.  The  first  can  be  re- 
moved as  soon  as  it  is  heated  through,  another  in  five  min- 
utes, another  in  ten  minutes,  or  at  whatever  intervals  the 
thought  and  experience  of  the  experimenter  shall  suggest, 
the  subsequent  growth  in  each  culture  showing  that  the 
endurance  of  the  organism  had  not  yet  been  exhausted. 
By  using  gelatin  and  pouring  each  culture  into  a  Petri  dish, 
and  subsequently  counting  the  colonies,  it  can  be  determined 
whether  many  or  only  a  few  of  the  organisms  in  a  culture 

300 


The  Thermal  Death-point  301 

possess  the  maximum  resisting  power.  To  determine  the 
percentage,  it  is  necessary  to  know  how  many  bacteria  were 
present  in  the  tubes  before  exposure  to  the  destructive 
temperature.  Approximately  the  same  number  can  be 
placed  in  each  tube  by  adding  the  same  measured  quantity 
of  a  fluid  culture  to  each. 

In  both  of  the  procedures  one  must  be  careful  that  the 
temperature  of  the  fluid  in  the  test-tube  is  identical  with  that 
of  the  water  in  the  bath.  A  sterile  thermometer  introduced 
into  an  uninoculated  tube  exposed  under  conditions  similar 
to  those  of  the  experiment  can  be  used  as  an  index  for  the 
others. 

Another  method  of  accomplishing  the  same  end  is  by  the 
use  of  Sternberg's  bulbs.  These  are  small  glass  bulbs  blown 
on  one  end  of  a  glass  tube,  drawn  out  to  a  fine  point  at  the 
opposite  end.  If  such  a  bulb  be  heated  so  that  the  air  is 
expanded  and  partly  driven  out,  its  open  tube,  dipped  into 
inoculated  bouillon,  will  in  cooling  draw  the  fluid  in,  so  as 
to  fill  it  one-third  or  one-half.  A  number  of  these  tubes  are 
filled  in  this  manner  with  a  freshly  inoculated  culture  medium 
and  then  floated,  tube  upward,  upon  a  water-bath  whose 
temperature  is  gradually  elevated,  the  bulbs  being  removed 
from  time  to  time  as  the  required  temperatures  are  reached. 
As  the  bulbs  are  already  inoculated,  all  that  is  necessary  is 
to  stand  them  aside  for  a  day  or  two,  and  observe  whether 
or  not  the  bacteria  grow,  determining  the  death-point  ex- 
actly as  in  the  other  case. 


CHAPTER   XVII. 

DETERMINATION  OF  THE  VALUE  OF  ANTISEP- 
TICS,  GERMICIDES,  AND  DISINFECTANTS* 

THE  student  must  bear  in  mind  that  an  antiseptic  is  a  sub- 
stance capable  of  restraining  the  growth  of  bacteria ;  a  germi- 
cide, one  capable  of  killing  them.  All  germicides  are  anti- 
septic in  dilute  solutions,  but  not  -all  antiseptics  are  germi- 
cides. Disinfectants  must  be  germicides. 

Antiseptics  are  chiefly  employed  for  purposes  of  preserva- 
tion, and  are  largely  used  in  the  industries  to  protect  organic 
substances  from  the  micro-organisms  of  fermentation  and 
decomposition.  The  problem  is  to  secure  a  satisfactory 
effect  with  the  addition  of  the  least  possible  preservative  in 
order  that  its  presence  shall  not  chemically  destroy  the  good 
qualities  of  the  substances  preserved.  In  the  case  of  foods 
it  becomes  necessary  to  use  preservatives  free  from  poisonous 
properties. 

Disinfectants  and  germicides  are  employed  for  the  purpose 
of  destroying  germs  of  all  kinds,  and  the  chief  problem  is  to 
secure  efficiency  of  action,  rather  than  to  endeavor  to  save 
on  the  reagent,  which  would  be  a  false  economy,  in  that  the 
very  object  desired  might  be  defeated. 

The  following  methods  of  determining  the  antiseptic  and 
germicidal  values  of  various  agents  can  be  elaborated  accord- 
ing to  the  extent  and  thoroughness  of  the  investigation  to 
be  made. 

I.  The  Antiseptic  Value. — Remembering  that  an  anti- 
septic is  a  substance  that  inhibits  bacterial  growth,  the  de- 
termination of  its  value  can  be  made  by  adding  varying 
quantities  of  the  antiseptic  to  be  investigated  to  culture 
media  in  which  bacteria  are  subsequently  planted.  It  is 
always  well  to  use  a  considerable  number  of  tubes  of  bouillon 
containing  varying  strengths  of  the  reagent  to  be  investi- 
gated. If  the  antiseptic  be  non-volatile,  it  may  be  added 
before  sterilization,  which  is  to  be  preferred ;  but  if  volatile, 
it  must  be  added  by  means  of  a  sterile  pipet,  with  the  greatest 

302 


The  Germicidal  Value  303 

precaution  as  regards  asepsis,  after  sterilization  and  imme- 
diately before  the  test  is  made.  Control  experiments — i.  e., 

bouillon  cultures  without  the  addition  of  the  antiseptic 

should  always  be  made. 

The  results  of  antiseptic  action  are  two:  retardation  of 
growth  and  complete  inhibition  of  growth.  As  the  inoculated 
tubes  containing  the  antiseptic  are  watched  in  their  devel- 
opment, it  will  usually  be  observed  that  those  containing 
very  small  quantities  develop  almost  as  rapidly  as  the  control 
tubes;  those  containing  more,  a  little  more  slowly;  those 
containing  still  more,  very  slowly,  until  at  last  there  comes  a 
time  when  the  growth  is  entirely  checked. 

Sternberg  points  out  that  the  following  conditions,  which 
must  be  avoided,  may  modify  the  results  of  experiment : 

1 .  The  composition  of  the  nutrient  media,  with  which  the 
antiseptic  may  be  incompatible  (as  bichloride  of  mercury 
and  albumin). 

2.  The  nature  of  the  test-organism,   no  two  organisms 
being  exactly  alike  in  their  susceptibility. 

3.  The  temperature  at  which  the  experiment  is  conducted, 
a  relatively  greater  amount  of  the  antiseptic  being  necessary 
at  temperatures  favorable  to  the  organism  than  at  tempera- 
tures unfavorable. 

4.  The  presence  of  spores  which  are  always  more  resistant 
than  the  asporogenous  forms. 

II.  The  Qermicidal  Value. — Koch's  original  method  of 
determining  this  was  to  dry  the  micro-organisms  upon  sterile 
threads  of  linen  or  silk,  and  then  soak  them  for  varying  lengths 
of  time  in  the  germicidal  solution.  After  the  bath  in  the 
reagent  the  threads  were  washed  in  clean,  sterile  water, 
transferred  to  fresh  culture  media,  and  their  growth  or  fail- 
ure to  grow  observed.  This  method  also  determines  the 
time  in  which  a  certain  solution  will  kill  micro-organisms,  so 
is  advantageous. 

Sternberg  suggested  a  method  by  which  the  dilution  nec- 
essary to  kill  the  bacteria  could  be  determined,  the  time 
remaining  constant  (two  hours'  exposure)  in  all  cases. 
"Instead  of  subjecting  test-organisms  to  the  action  of  the 
disinfecting  agent  attached  to  a  silk  thread,  a  certain  quan- 
tity of  a  recent  culture — usually  5  c.c. — is  mixed  with  an 
equal  quantity  of  a  standard  solution  of  the  germicidal 
agent,  .  .  .  and  after  two  hours'  contact  one  or  two 
loopfuls  are  transferred  to  a  suitable  nutrient  medium  to 
test  the  question  of  disinfection." 


3°4 


Value  of  Antiseptics 


A  very  simple  and  popular  method  of  determining  the 
germicidal  value  is  to  make  a  series  of  dilutions  of  the  re- 
agent to  be  tested ;  add  to  each  a  small  quantity  of  a  fresh 
liquid  culture,  and  at  varying  intervals  of  time  transfer  a 
loopful  to  fresh  culture  media.  By  a  little  ingenuity  this 


...A 


Same  rod  immersed  in  broth  after  exposure 
to  disinfectant. 

Fig.  88.— Glass  rod  in  test-tube,  for  use  in  testing  disinfectants. 
Tube  6  in.  by  f  in. ;  rod  9  in.  by  £  in.  Ring  marked  with  diamond  1  in. 
from  lower  end,  to  show  upper  limit  of  area  on  which  the  organisms 
are  dried.  After  exposure  the  rod  is  placed  in  a  similar  tube  con- 
taining broth,  to  test  development,  a,  Cotton  plug  wrapped  around 
glass  rod ;  b,  broth ;  c,  gummed  label  on  handle  of  rod,  for  indenti- 
fication;  d,  ring  marked  by  diamond;  e,  dried  organisms. 


method  may  be  made  to  yield  information  as  to  both  time 
and  strength. 

Hill  *  has  suggested  a  convenient  method  of  handling  the 
cultures,  which  are  dried  upon  the  ends  of  sterile  glass  rods 
and  can  then  be  transferred  from  one  solution  to  another  or 
otherwise  manipulated  (see  Fig.  88). 

*  "Public  Health,"  vol.  xxiv,  p.  246. 


The  Germicidal  Value  305 

One  of  the  best  methods  for  testing  the  germicidal  value 
of  solutions  is  that  suggested  by  Rideal  and  Walker.*  To 
use  it,  it  is  necessary  to  proceed  with  strict  attention  to 
details. 

The  advantage  of  this  method  is  that  it  expresses  the 
germicidal  value  in  terms  of  carbolic  acid,  as  the  "  carbolic 
acid  coefficient,"  and  thus  makes  possible  an  accurate  com- 
parison of  germicide  with  germicide. 

The  test  culture  should  be  grown  in  bouillon  made  accord- 
ing to  the  same  formula  with  exactly  the  same  reaction. 
The  cultures  should  be  grown  in  the  thermostat  at  37°  C. 
for  just  twenty-four  hours,  and,  in  order  that  they  should 
contain  no  clumps  of  bacteria,  should  be  carefully  filtered 
through  cotton-wool  or  glass  wool  just  before  using.  The 
transfer  of  the  bacteria  from  the  filtered  culture  to  the  diluted 
germicide  solutions  is  made  with  a  large  platinum  loop, 
about  6  mm.  in  diameter,  slightly  bent  like  a  spoon,  so  as  to 
take  up  a  large  drop.  Three  drops  are  carried  from  the 
culture  into  the  germicide  solution,  which,  no  matter  what 
its  dilution,  should  be  of  5  c.c.  volume,  with  great  care  that 
the  walls  and  mouth  of  the  tube  are  not  touched.  The  tube 
is  then  well  shaken  from  side  to  side,  so  as  to  distribute  the 
bacteria  throughout  the  solution,  and  the  tube  set  aside. 
At  intervals  of  one,  three,  five,  ten,  and  fifteen  minutes 
respectively,  three  drops  of  the  fluid  are  transferred  with 
the  same  platinum  loop  to  fresh  bouillon  tubes.  Failure 
to  grow  upon  such  transplantation  shows  that  the  bacteria 
have  been  killed. 

Gaseous  Disinfection. — If  the  germicide  to  be  studied  be 
a  gas,  as  in  the  case  of  sulphurous  acid  or  formaldehyd,  a 
different  method  must,  of  course,  be  adopted. 

It  may  be  sufficient  to  place  a  few  test-tube  cultures  of 
various  bacteria,  some  with  plugs  in,  some  with  plugs  out, 
in  a  closed  chamber  in  which  the  gas  is  evolved.  The  germi- 
cidal action  is  shown  by  the  failure  of  the  cultures  to  grow 
upon  transplantation  to  fresh  culture  media.  This  crude 
method  may  be  supplemented  by  an  examination  of  the  dust 
of  the  room.  Pledgets  of  sterile  cotton  are  rubbed  upon  the 
floor,  washboard,  or  any  dust-collecting  surface  present,  and 
subsequently  dropped  into  culture  media.  Failure  of  growth 
under  such  circumstances  is  very  certain  evidence  of  good 
disinfection.  These  tests  are,  however,  very  severe,  for  in 
20 


306  Value  of  Antiseptics 

the  cultures  there  are  immense  numbers  of  bacteria  in  the 
deeper  portions  of  the  bacterial  mass  upon  which  the  gas  has 
no  opportunity  to  act,  and  in  the  dust  there  are  many  spor- 
ogenous  organisms  of  extreme  resisting  power.  Failure  to 
kill  all  the  germs  exposed  in  such  manner  is  no  indication 
that  the  vapor  cannot  destroy  all  the  ordinary  pathogenic 
organisms. 

A  more  refined  method  of  making  the  tests  consists  in 
saturating  strips  of  blotting-paper,  absorbent  cotton,  various 
fabrics,  etc.,  with  cultures  and  exposing  them,  moist  or  dry, 
to  the  action  of  the  gas.  Such  materials  are  best  made 
ready  in  Petri  dishes,  which  are  opened  immediately  before 
and  closed  immediately  after  the  experiment.  If,  when 
transferred  to  fresh  culture  media,  the  exposed  objects  fail 
to  give  any  growth,  the  disinfection  has  been  thorough.  If 
the  penetrating  power  of  a  gas,  such  as  formaldehyd,  is  to 
be  tested,  it  can  be  done  by  inclosing  the  infected  paper  or 
fabrics  in  envelopes,  boxes  perforated  with  small  holes, 
tightly  closed  pasteboard  boxes,  and  by  wrapping  them  in 
towels,  blankets,  mattresses,  etc. 

Easier  of  execution,  but  rather  more  severe,  is  a  method 
in  which  cover-glasses  are  employed.  A  number  of  them 
are  sterilized,  spread  with  cultures  of  various  bacteria,  al- 
lowed to  dry,  and  then  exposed  to  the  gas  as  long  as  re- 
quired. They  are  subsequently  dropped  into  culture  media 
to  permit  the  growth  of  the  organisms  not  destroyed. 

Animal  experiments  may  also  be  employed  to  determine 
whether  or  not  a  germ  that  has  survived  exposure  to  the 
action  of  reagents  has  its  pathogenic  power  destroyed.  An 
excellent  example  of  this  is  seen  in  the  case  of  the  anthrax 
bacillus,  a  virulent  form  of  which  will  kill  rabbits,  but  after 
being  grown  in  media  containing  an  insufficient  amount  of  a 
germicide  to  kill  it  will  often  lose  its  rabbit-killing  power, 
though  still  able  to  fatally  infect  guinea-pigs,  or  may  lose  its 
virulence  for  both  rabbits  and  guinea-pigs,  though  still  able 
to  kill  white  mice. 


CHAPTER   XVIII 

THE  PHAGOCYTIC  POWER  OF  THE  BLOOD  AND 
THE  OPSONIC  INDEX. 

FROM  the  time  that  Metschnikoff  connected  the  phenomena 
of  phagocytosis  with  those  of  immunity  until  1902,  there 
was  no  recognized  technic  for  the  observation  and  com- 
parison of  the  bacteria-consuming  and  bacteria-destroying 
power  of  the  cells.  In  1902  Leishman*  suggested  the 
following  simple  technic: 

A  thin  suspension  of  bacteria  in  normal  salt  solution  is 
mixed  with  an  equal  volume  of  blood  by  drawing  in  and 
out  of  a  capillary  tube,  then  dropped  upon  a  clean  slide, 
covered  carefully,  placed  in  a  moist  chamber,  and  incubated 
at  37°  C.  for  a  half  hour.  The  cover  is  then  slipped  off 
carefully,  as  in  making  blood-spreads,  dried,  stained,  and 
the  number  of  bacteria  in  each  of  20  leukocytes  counted 
and  averaged.  For  comparison  with  the  normal  the  patient's 
blood  and  normal  blood  are  simultaneously  examined. 

This  was  greatly  improved  by  Wright  and  Douglas,  f  the 
accuracy  of  whose  methods  enabled  them  to  discover  the 
"opsonins, "  work  out  the  "opsonic  index,"  and  formulate 
methods  by  which  sufficiently  accurate  observations  could 
be  made  for  controlling  the  specific  treatment  of  infectious 
diseases. 

The  opsonic  theory  teaches  that  the  leukocytes  are 
disinclined  to  take  up  bacteria  unless  they  are  prepared 
for  consumption  or  phagocytosis  by  contact  with  certain 
substances  in  the  serum  that  in  some  manner  modify  them. 
This  modifying  substance  is  the  opsonin  (opsono,  I  cater  to, 
I  prepare  for). 

To  make  a  test  of  the  opsonic  value  of  the  blood  it  is 
necessary  to  prepare  the  following : 

A  uniform  suspension  of  bacteria. 

A  suspension  in  salt  solution  of  washed  leukocytes. 

The  serum  to  be  tested. 

A  normal  serum  for  comparison. 

*  "British  Medical  Journal,"  1902,  I,  Jan.  n,  p.  73. 
t  "Proc.  Royal  Soc.  of  London,"  1904,  LXXXII,  p.  357. 
307 


308         The  Phagocytic  Power  of  the  Blood 

The  Bacterial  Suspension. — This  is  prepared  like  the 
similar  suspensions  used  for  determining  agglutination,  but 
with  greater  care,  since  the  bacteria  taken  up  by  the  cor- 
puscles are  to  be  counted,  and  any  variation  in  the  number 
of  bacteria  with  which  they  come  into  contact  may  modify 
the  count.  It  is  also  necessary  to  avoid  all  clumps  of  bac- 
teria for  the  same  reason. 

The  culture  is  best  grown  upon  agar-agar  for  twelve  to 
twenty-four  hours,  the  bacteria  in  young  cultures  being  more 
easy  to  separate  than  those  in  old  cultures.  Such  a  culture 
may  be  taken  up  in  a  platinum  loop,  transferred  to  a  test- 
tube  containing  some  0.85  per  cent,  sodium  chloride  solution, 


Fig.  89. — Grinding  bacteria  (Miller). 

and  gently  rubbed  upon  the  glass  just  above  the  fluid, 
allowing  the  moistened  and  mixed  bacterial  mass  to  enter 
the  fluid  little  by  little. 

If  the  culture  be  older  or  of  a  nature  that  will  not  separate 
in  this  manner  (tubercle  bacillus),  it  may  be  necessary  to 
rub  it  between  two  glass  plates  (Fig.  89),  or  in  a  small  agate 
mortar  with  a  drop  or  two  of  salt  solution,  other  drops  being 
added  one  at  a  time,  until  a  homogeneous  suspension  is 
secured.  Such  clumps  of  bacteria  as  may  remain  in  the 
suspension  are  easily  removed  by  whirling  for  a  few  seconds 
in  a  centrifuge. 

The  next  step  is  the  standardization  of  the  suspension. 
Wright  recommends  for  this  purpose  and  for  the  standard- 
ization of  the  bacterio-vaccines  that  the  number  of  bacteria 
shall  actually  be  counted.  This  he  does  by  mixing  one  part 
of  the  bacterial  suspension  with  an  equal  volume  of  normal 


The  Bacterial  Suspension 


309 


blood  and  three  volumes  of  physiological  salt  solution. 
After  thorough  mixing  a  smear  is  made  upon  a  slide,  the 
smear  stained,  and  the  number  of  bacteria  and  corpuscles  in 
successive  fields  of  the  micro- 
scope counted  until  at  least 
200  red  blood-corpuscles  have 
been  enumerated.  As  the 
number  of  red  corpuscles  per 
cubic  millimeter  of  blood  is 
5,000,000,  the  number  of  bac- 
teria per  cubic  centimeter  can 
be  determined  from  the  re- 
sults of  the  counting  by  a 
simple  arithmetical  process. 
To  facilitate  the  counting  the 
eye-piece  of  the  microscope  is  prepared  by  the  introduction 
of  a  diaphragm  shown  in  Fig.  90.  The  prepared  suspen- 
sion must  usually  be  greatly  diluted  before  using,  but  the 


Fig.  90. — Diaphragm  of  eye- 
piece showing  hairs  in  position 
(Miller). 


Fig.  91. — Photomicrograph  showing  cross-hairs,  bacteria,  and  red  blood- 
corpuscles  (Miller). 


reduction  of  bacteria  is,  of  course,  easily  calculated.  It  re- 
quires experience  to  determine  the  appropriate  number  of 
bacteria  to  be  employed.  When  this  is  once  determined, 


310         The  Phagocytic  Power  of  the  Blood 

future  manipulations  are  made  easy,  because  one  first  makes 
his  suspension,  then  enumerates  the  bacteria,  and  having 
determined  their  number,  immediately  arrives  at  the 
appropriate  concentration  by  dilution  (Fig.  91). 


Fig.  92. — The  nephelometer ;  an  instrument  used  for  standardizing 
bacterial  suspensions  used  for  the  opsonic  test  or  for  vaccines,  by  com- 
parison with  precipitate  of  barium  sulphate. 

The  writer*  believes  that  equally  accurate  results  can  be 
attained  by  means  of  a  simple  instrument  which  he  has 
called  a  "  nephelometer"  (Fig.  92).  By  the  use  of  this 


-  93-  —  Collecting  blood  for  corpuscles  (Miller). 


instrument  one  arrives  at  the  appropriate  dilution  of  the 

bacterial  suspension  by  comparison  with  a  tube  containing 

*  "  Jour.  Amer.  Med.  Assoc.,"  Oct.  5,  1907,  XLIX,  p.  1176. 


The  Washed  Leukocytes 


barium  sulphate  precipitate.  By  careful  comparison  of  the 
suspension  with  the  standard  tube,  and  the  addition  of 
more  of  the  salt  solution  or  of  the  bacteria,  as  may  be 
required,  one  can  arrive  at  fairly  uniform  results  in  a  few 
moments. 

The  Washed  Leukocytes. — It  is  not  necessary  to  have 
the  leukocytes  free  from  admixture  with  the  erythrocytes, 
but  it  is  necessary  to  have  large  numbers  of  them.  They 
are  collected  by  citrating  the  blood  so  as  to  prevent  coagula- 
tion, and  then  separating  the  citrated  plasma  from  the 
corpuscles  by  centrifugalization. 

The  hands  of  the  patient  are  washed,  and  a  piece  of  elastic 
rubber  tubing  or  some  other  convenient  fillet  wound  about 
the  thumb  or  a  finger  to  produce  venous 
congestion.     With  a   convenient  lancet 
(Wright  uses  a  pricker  made  by  drawing 
a  bit  of  glass  tubing  or  a  glass  rod  to  a 
fine  point  in  the  flame)  a  prick  is  made 
about  a  quarter  inch  from  the  root  of 
the  nail  (Fig.  93).     From  this  the  blood 
is  permitted  to  flow  into  small  test-tubes 
previously  filled  about  three-fourths  with 
1.5   per  cent,    sodium    citrate    solution. 
The  blood  and  citrate  solution  are  mixed, 
and  the  tubes   placed   in  a  centrifuge, 
balanced,  and   centrifugalized  until  the 
corpuscles  are  collected  at  the  bottom 
of   the   tube    (Fig.    94).     The    citrated 
plasma  is  now  withdrawn  and  replaced 
with  0.85  per  cent,  sodium  chloride  solu- 
tion, through  which  the  corpuscles  are 
distributed  by  shaking.     The  tubes  are 
now  again  centrifugalized  until  the  corpuscles  are  collected, 
when  the  saline  is  removed  carefully,  the  last  drop  from 
the  back  of  the  meniscus  (Fig.  95).      In  the  corpuscular 
mass  that  remains  the  leukocytes  form  a  thin  creamy  layer 
on  the  top. 

The  serum  to  be  tested  and  the  normal  serum  for 
comparison  are  secured  in  the  same  manner,  the  former 
from  the  patient,  the  latter  from  the  operator.  As  it  is 
advisable  to  wound  the  patient  but  once,  the  tube  for 
obtaining  the  serum  should  be  filled  at  the  same  time  that 
the  citrated  blood  is  taken. 


Fig.  94. — Tube  of 
blood  and  citrate 
solution  before  and 
after  centrifugaliz- 
ing  (Miller). 


312         The  Phagocytic  Power  of  the  Blood 

The  blood  to  furnish  the  serum  is  taken  in  a  small  bent 
tube  shown  in  Fig.  96. 

The  blood  flowing  from  the  puncture  is  allowed  to  flow 
into  the  bent  end  of  the  tube,  into  which  it  enters  by  cap- 
illary attraction  and  from  which  it  descends  to  the  body  of 
the  tube  by  gravity.  At  least  one  cubic  centimeter  of  the 
blood  is  required  to  furnish  the  serum.  The  ends  of  the 
tube  are  closed  in  the  flame  and  the  tube  stood  in  the  ther- 
mostat for  fifteen  to  thirty  minutes.  Coagulation  takes 
place  almost  immediately,  and  the  serum  usually  separates 
quickly.  If  it  does  not  do  so,  Wright  recommends  hanging 


Fig.  95. — Removing  last  drops  of  saline  solution  (Miller). 

the  curved  arm  of  the  tube  over  the  centrifuge  tube  and 
whirling  it  for  a  moment  or  two,  when  the  clot  is  driven  into 
the  straight  arm  of  the  tube  and  the  clear  serum  appears 
above.  The  tube  is  then  cut  with  a  file  so  that  the  serum  can 
be  removed  when  needed.  Mixing  the  factors  concerned  in 
the  test  is  a  matter  that  requires  practice  and  a  steady  hand. 
It  is  best  done,  as  recommended  by  Wright,  in  a  capillary 
tube  controlled  by  a  rubber  bulb  (Fig.  97).  The  object 
of  the  experimenter  is  to  take  up  into  this  pipette  equal 
quantities  of  the  creamy  layer  of  blood-corpuscles,  of  the 
blood-serum,  and  of  the  bacterial  suspension.  Wright  first 
makes  a  mark  with  a  wax  pencil  about  i  centimeter  from  the 
end  of  the  capillary  tube.  He  first  draws  up  the  leukocytic 


The  Washed  Leukocytes 


layer  of  blood-corpuscles  to  this  mark,  then  removing  the 
tube,  permits  the  column  to  as- 
cend a  short  distance.  Next  he 
draws  up  the  bacterial  suspension 
to  the  same  point,  withdraws  the 
tube,  and  permits  the  column  to 
ascend;  then  draws  up  the  serum 
to  be  taken  to  the  same  point; 


Fig.  96.— Special  blood  pipette  (Miller). 

thus  in  the  same  capillary  tube 
he  has  three  equal  volumes  of 
three  different  fluids,  separated  by 
bubbles  of  air.  It  is  next  neces- 
sary to  mix  these,  which  is  done 
by  repeatedly  expelling  them  upon 
a  clean  glass  slide,  and  redrawing 
them  into  the  tube,  as  shown  in 
Fig.  98.  After  thus  being  thor- 
oughly mixed,  the  fluid  is  once 
more  permitted  to  enter  the  capil- 
lary tube  and  come  to  rest  there. 


Fig.  97. — Opsonizing  pi- 
pette containing  blood-cor- 
puscles, bacterial  emulsion, 
and  blood-serum  (Miller). 

The  end  is  now  sealed  in 


314         The  Phagocytic  Power  of  the  Blood 

a  flame,  the  rubber  bulb  removed  and  the  tube  placed  in  a 
thermostat,  or  in  case  much  work  of  the  kind  is  being  done, 
to  an  opsonizing  incubator  (Fig.  99)  in  which  the  temperature 
is  not  modified  by  opening  and  closing  the  doors.  The  tube 
remains  in  the  incubating  apparatus  at  37°  C.  for  fifteen 
minutes  (some  use  twenty,  some  thirty,  minutes  as  their 
standard),  is  then  removed,  whirled  about  its  long  axis 
between  the  thumbs  and  fingers  a  few  times  to  mix  the 
contents  from  which  the  corpuscles  have  sedimented,  its 


Fig.  98.- 


-Mixing  liquids  by  repeatedly  expelling  on  to  slide  and  redraw- 
ing into  pipette  (Miller). 


end  is  broken  off,  and  a  good-sized  drop  is  allowed  to  escape 
upon  a  perfectly  clean  glass  slide  and  spread  over  its  surface. 

The  spreading  is  a  matter  of  some  importance,  as  an  even 
distribution  of  the  leukocytes  is  desirable.  The  capillary 
tube  from  which  the  drop  has  escaped  will  form  a  good 
spreader  if  laid  flat  upon  the  glass  and  drawn  along,  but 
the  edge  of  another  slide  is  better,  and  in  distributing  the 
fluid,  it  is  better  to  push  than  to  pull  it  with  the  end  of  the 
slide,  rather  than  its  side. 

Miller*  says  that  "a  good  smear  should  be  uniform  in 
consistency  and  most  of  the  leukocytes  should  be  found 
along  the  edges  and  at  the  end.  For  convenience  in  count- 
ing, it  is  well  to  have  the  smear  terminate  abruptly  and  not 
be  drawn  out  into  threads  or  irregular  forms." 

*  "Therapeutic  Gazette,"  March  15,  1907. 


The  Washed  Leukocytes 


This  mixing,  incubating,  and  spreading  is  done  twice — 
once  with  the  serum  of  the  patient,  and  once  with  the  normal 
serum  of  the  operator.  The  technic  is  the  same  each  time. 


Fig-  99- — A  small  incubator  of  special  design  for  opsonic  work  (Miller) . 

In  order  that  the  enumeration  of  the  bacteria  taken  up  by 
the  leukocytes  can  be  accomplished,  it  is  next  necessary  to 
stain  the  blood  smears.     This  can  be  done  by  any  method 
that  will  demonstrate  both 
the  bacteria  and  the  cells. 
For      staphylococci      and 
similar  organisms,    Irish- 
man's stain,  Jenner's  stain, 
or  J.    H.   Wright's   stains 
are  appropriate.    Marino's 
stain,     recommended     by          FiS-  100.— The  smear  (Miller). 
Levaditi,*  gives  beautiful 

results.     For  the  tubercle  bacillus  the  spreads  may  be  stained 

with    carbol-fuchsin    and    counterstained   with    methylene- 

*  "Ann.  de  1'Inst.  Pasteur,"  1904,  xvm,  p.  761. 


3i 6        The  Phagocytic  Power  of  the  Blood 

blue,  or  perhaps  better  with  gentian  violet  and  counter- 
stained  with  Bismarck  brown  or  vesuvin. 

The  final  step  in  the  process  is  the  enumeration  of  the 
bacteria  in  the  corpuscles  by  averaging  the  number  taken 
up  by  the  cells.  Only  typical  polymorphonuclear  cells 
should  be  selected  for  staphylococcic  cases,  and  separate 
averages  made  for  polymorphonuclear  and  mononuclear 
cells  in  tubercle  bacillus  cases.  It  is  best  to  follow  cer- 
tain routine  methods  of  enumeration.  Some  who  content 
themselves  with  a  count  of  the  number  of  bacteria  in  20  cells, 
secure  less  accurate  results  than  those  who  count  50  cells. 
It  is  usually  best  to  count  one-third  of  the  cells  in  the  central 
portion  of  the  spread,  one- third  at  the  edge,  and  one-third 
at  the  end.  In  each  portion  no  other  selection  of  cells  should 
be  made  than  the  elimination  of  other  than  polymorpho- 
nuclear cells  and  the  elimination  of  all  crushed  or  injured 
cells ;  the  others  should  be  taken  one  after  the  other,  as  they 
are  brought  into  the  field  with  the  mechanical  stage.  After 
the  bacteria  included  in  each  of  the  accepted  number  of 
cells  selected  as  the  standard  has  been  enumerated,  an 
average  is  struck. 

The  "opsonic  index"  is  determined  by  dividing  the 
average  number  in  the  patient's  serum  preparation  by  the 
average  in  the  normal  serum  preparation. 

Irishman's*  studies  of  the  phagocytic  power  of  the  blood 
show  that  in  cases  of  furunculosis,  etc.,  with  each  recru- 
descence of  boils,  there  is  a  marked  diminution  of  the  pha- 
gocytic power  of  the  blood,  and  with  each  improvement, 
a  marked  increase. 

McFarland  and  1'Englet  found  by  an  examination  of  the 
blood  of  24  supposedly  healthy  students  and  laboratory 
workers  that  it  was  possible  to  prejudge,  by  the  phagocytic 
activity  of  the  cells,  the  past  occurrence  of  suppuration  and 
present  liability  to  it. 

Wright  and  Douglas  use  the  opsonic  index  as  a  guide  to 
the  specific  therapy  of  the  infectious  diseases.  If  the 
opsonic  index  is  low  they  believe  bacterio-vaccination  is 
indicated.  In  its  administration,  however,  care  must  be 
taken  to  administer  a  counted  number  of  bacteria,  and  to 
make  frequent  opsonic  estimations  to  determine  the  good 
or  ill  effects  accomplished.  Thus,  the  administration  is 

*  "Lancet,"  1902,  i,  p.  73. 
f  "Medicine,"  April,  1906. 


The  Washed  Leukocytes  317 

always  followed  by  a  temporary  diminution  (negative  phase) 
of  the  opsonic  index,  soon  followed,  if  the  dose  be  not  too 
large,  by  a  marked  increase  (positive  phase).  It  is  sup- 
posed, upon  theoretic  grounds,  and  proved  by  practical 
experience,  that  the  increase  of  phagocytic  activity  brings 
about  improvement.  The  care  of  the  operator  should  be 
to  avoid  giving  so  large  a  dose  of  the  vaccine  that  the  nega- 
tive phase  will  be  so  long  continued  that  harm  instead  of  good 
may  be  achieved. 

Although  Wright  is  said  to  cling  to  the  study  of  the  opsonic 
index  as  a  guide  to  bacterio-vaccination  and  the  resulting 
degree  of  immunity,  the  greater  number  of  workers  have 
abandoned  it  upon  grounds  which  the  writer  long  ago  ex- 
pressed— "  that  the  estimation  of  the  value  of  bacterio- 
vaccination  by  means  of  the  opsonic  index  was  a  very  com- 
plicated way  of  finding  out  very  little." 


CHAPTER   XIX. 

THE   WASSERMANN    REACTION   FOR   THE 
DIAGNOSIS   OF   SYPHILIS. 

THIS  now  popular  and  fairly  reliable  method  for  assisting 
in  the  diagnosis  of  atypical  syphilitic  infections  was  devised 
by  Wassermann,  Neisser,  and  Bruck.*  It  is  a  method  of 
making  the  diagnosis  of  syphilis  by  demonstrating  in  the 
blood  (cerebrospinal  fluid,  milk,  or  urine)  of  the  patient  a 
complement-fixing  substance  (antibody?)  not  present  in  nor- 
mal blood. 

The  test  is  twofold:  (i)  A  combination  of  syphilitic 
antigen,  complement,  and  suspected  serum.  (2)  A  subse- 
quent addition  to  the  mixture  of  blood-corpuscles  and  hemo- 
lytic  amboceptor.  If  the  suspected  serum  contain  the 
syphilitic  antibody  the  antigen  and  complement  unite  with 
it,  and  the  complement  being  thus  "  fixed,"  no  hemolysis 
can  take  place  upon  the  subsequent  addition  of  the  blood-cor- 
puscles and  hemolytic  serum.  If,  on  the  other  hand,  the  sus- 
pected serum  contain  no  antibody,  the  complement  cannot  be 
fixed,  and  is,  therefore,  free  to  act  upon  the  subsequently 
added  blood-corpuscles  in  the  presence  of  the  hemolytic 
serum,  and  hemolysis  results. 

It  is  thus  seen  that  the  first  test  is  made  for  the  purpose  of 
fixing  the  complement,  and  the  second  for  the  purpose  of 
finding  out  whether  it  has  been  fixed  or  not. 

It  is  quite  clear  that  such  a  test  is  very  delicate,  and  can 
only  be  successful  when  executed  with  great  precision  and  with 
reagents  or  factors  titrated,  so  that  their  exact  value  may  be 
known. 

CONSIDERATION  OF  THE  REAGENTS  EMPLOYED. 

I.  For  the  first,  or  fixation,  test  it  is  necessary  to  bring 
together — 

Syphilitic  antigen. 
Serum  to  be  tested. 
Complement. 

*  "Deutsch.  Med.  Wochenschr.,"  1906,  No.  19. 


The  Syphilitic  Antigen  319 

(i)  The  Syphilitic  Antigen. — It  was  supposed  by  Wasser- 
mann,  Neisser,  and  Bruck,  who  first  devised  the  test,  that 
the  syphilitic  antigen  must  contain  the  essential  micro- 
organisms of  syphilis.  No  method  for  the  cultivation  of  Tre- 
ponema  pallidum  having  at  that  time  been  devised,  cultures 
of  the  specific  micro-organism  could  not  be  employed. 
Histologists  had,  however,  shown  that  greater  numbers  of 
the  organisms  were  to  be  found  in  the  livers  of  the  congeni- 
tally  syphilitic  stillborn  infants  than  anywhere  else.  With 
the  purpose,  therefore,  of  securing  the  greatest  possible  num- 
ber of  micro-organisms  for  the  antigenic  function,  such  livers 
were  used.  The  tissue,  having  been  cut  into  small  fragments, 
was  spread  out  in  Petri  or  other  appropriate  dishes  and  dried, 
and  the  fragments  rubbed  to  a  fine  powder  with  a  mortar  and 
pestle.  Such  a  powder  can  be  kept  indefinitely  in  an  exsic- 
cator over  calcium  chlorid  if  placed  where  it  is  cool  and  dark. 
When  the  powder  is  to  be  used,  0.5  gin.  is  extracted  either  at 
room  temperature  or  in  the  ice-box  with  25  c.c.  of  95  per  cent, 
alcohol  for  twenty-four  hours,  filtered  through  paper,  and  the 
filtrate  used  in  quantities  later  to  be  mentioned. 

Instead  of  drying  the  liver  tissue,  pulverizing,  and  then 
extracting  it,  many  investigators  now  prefer  to  cut  it  up,  rub 
it  into  a  uniform  paste  with  a  mortar  and  pestle,  and  add  5 
volumes  of  95  per  cent,  or  absolute  alcohol,  with  which  the 
paste  is  thoroughly  macerated  and  shaken  many  times  or  in 
a  shaking  machine.  The  alcohol  may  then  be  filtered  off, 
or  may  be  permitted  to  remain  upon  the  sedimented  liver 
tissue  remnants,  and  the  clear  supernatant  fluid  pipe  ted  off 
and  diluted,  at  the  time  of  employment,  with  the  isotonic 
sodium  chlorid  solution.  When  this  alcoholic  extract  is 
added  to  the  salt  solution  a  turbidity  occurs,  but  this  must 
not  be  filtered  out,  as  it  consists  of  the  lipoids  or  other  sub- 
stances in  the  extract  that  are  essential  to  the  test,  and  the 
quantity  of  the  cloudy  fluid  in  the  final  mixtures  is  so  small 
as  not  in  any  way  to  interfere  with  the  results.  The  small 
amount  of  alcohol  in  the  diluted  extract  is  negligible  and  has 
no  influence  upon  the  reagents  used  for  the  test. 

The  mention  of  the  lipoids  now  brings  us  to  the  point  where 
it  seems  advisable  to  state  that  one  of  the  most  interesting 
facts  about  the  Wassermann  reaction  is  that  its  theoretic 
basis  was  founded  upon  the  erroneous  assumption  that  the 
essential  antigenic  substance  consisted  of  the  whole  or  frag- 
mented treponemata  in  the  liver  extract.  The  method 


320   Wassermann  Reaction  for  Diagnosis  of  Syphilis 

scarcely  began  to  meet  with  practical  application,  however, 
before  it  was  discovered  that  the  active  antigenic  substance 
was  soluble  in  alcohol,  was  present  in  other  than  syphilitic 
livers,  and  could  be  extracted  not  only  from  human  tissues, 
but  also  from  dogs'  livers  and  from  guinea-pigs'  hearts. 
Forges  and  Meier,  indeed,  found  that  lecithin  could  play  the 
role  of  syphilitic  antigen,  and  Leviditi  and  Yamanonchi 
place  sodium  glycocholate,  sodium  taurocholate,  protogon, 
and  cholin  among  those  bodies  capable  of  acting  as  syphilitic 
antigens,  and  Noguchi  goes  so  far  from  the  original  that  he 
regularly  employs  an  extract  of  the  normal  guinea-pig's  heart 
as  the  antigen  to  be  employed  in  his  modification  of  the  test. 

These  discoveries  now  make  it  clear  that  the  complement 
fixation  that  takes  place  in  syphilis  is  not  identical  with  that 
of  the  Bordet-Gengou  reaction,  in  which  it  had  its  beginning. 
Happily,  however,  the  error  does  not  destroy  the  usefulness 
of  the  method  for  diagnosis. 

The  probable  nature  of  the  reaction  will  be  described  below. 
For  the  present  we  must  be  content  to  follow  the  beaten  path, 
and  for  this  purpose  will  use  the  congenitally  syphilitic  liver 
extract  as  the  antigen,  preparing  it  as  described  above. 

(2)  The. Serum  to  be  Tested. — Wassermann,  Neisser,  and 
Bruck  at  first  employed  the  cerebrospinal  fluid,  but  now  the 
blood-serum  of  the  suspected  patient  is  almost  universally 
used.  As  is  usual  with  antibodies,  the  substances  engaging 
in  the  complement-fixation  test  are  widely  distributed 
throughout  the  body,  and  reach  the  cerebrospinal  fluid,  the 
milk,  the  urine,  and  the  other  body  fluids  through  the  blood, 
in  which  it  exists  in  greatest  concentration.  The  blood  is, 
moreover,  readily  obtainable  for  study,  which  is  another 
reason  it  is  at  present  used  for  making  the  test  under  all 
ordinary  circumstances.  Some  workers  who,  like  Noguchi, 
work  with  very  small  quantities  of  the  reagents,  secure 
the  blood  by  obstructing  the  venous  circulation  of  the 
thumb  or  of  a  finger  by  means  of  a  rubber  band  (see  di- 
rections for  obtaining  the  blood  for  making  the  opsonic 
index),  but  the  greater  number  prefer  to  obtain  it  by  in- 
troducing a  large  hypodermic  needle  into  one  of  the  veins 
near  the  bend  of  the  elbow.  The  arm  above  the  elbow 
is  compressed  by  a  fillet,  as  though  for  the  purpose  of 
performing  phlebotomy,  and  a  conspicuous  vein  selected 
for  the  purpose.  The  skin  is  first  carefully  washed,  then 
treated  with  tincture  of  iodin.  If  the  patient  is  nervous,  a 


The  Complement  321 

momentary  spraying  with  chlorid  of  ethyl  will  make  the 
operation  entirely  painless.  Some  prefer  to  use  the  iodin 
without  the  preliminary  washing,  believing  that  soap 
makes  it  difficult  for  the  iodin  to  effect  satisfactory  dis- 
infection of  the  skin.  The  sterilized  needle  is  thrust  into 
the  vein,  care  being  taken  that  the  vein  is  not  too  com- 
pressed and  the  point  of  the  needle  thrust  entirely  through 
instead  of  into  it.  From  15  to  25  c.c.  of  blood  may  be  with- 
drawn into  a  large  syringe  or  may  be  allowed  to  flow  into  a 
sterile  test-tube.  The  blood,  however  secured,  is  permitted 
to  coagulate  and  the  clear  serum  removed  by  a  pipet,  or  the 
clotted  blood  is  placed  in  a  centrifuge  tube  and  whined,  so 
that  clear  serum  is  secured  in  a  few  minutes. 

As  normal  human  blood-serum,  when  fresh,  contains  a 
certain  amount  of  complement  which  would  interfere  with 
the  success  of  the  experiment,  the  serum  is  next  placed  in  a 
test-tube  and  kept  in  a  water-bath  between  55°  to  58°  C.  for 
a  half -hour.  This  degree  of  heat  destroys  the  complement 
and  leaves  the  complement-fixing  substance  uninjured.  The 
serum  is  now  ready  for  use. 

(3)  The  Complement. — The  complement  universally  em- 
ployed is  contained  in  the  blood  of  a  healthy  adult  guinea-pig. 
To  obtain  it  a  piece  of  cotton  moistened  with  ether  or  chloro- 
form is  held  to  the  guinea-pig's  nose  until  it  becomes  uncon- 
scious, when  the  head  is  forcibly  extended  and  a  longitudinal 
incision  made  through  the  skin  of  the  neck.  The  skin  is  then 
drawn  back  between  the  finger,  on  the  one  side,  and  the  thumb, 
on  the  other  side,  of  the  operator's  left  hand,  while,  with  a 
sharp  knife  held  in  the  right  hand,  he  cuts  through  all  of  the 
tissues  of  the  neck  down  to  the  spinal  column  and  thus  opens 
both  carotid  arteries.  The  spurting  blood  is  caught  in  a 
sterile  Petri  dish  and  the  animal  permitted  to  bleed  to  death. 
The  blood  soon  coagulates  when  undisturbed,  and  in  a  short 
time  clear  serum  exudes  from  the  clot.  As,  however,  the 
complement  seems  to  be  at  least  in  part  derived  from  the 
corpuscles,  the  serum  should  not  be  removed  as  soon  as  it 
forms,  but  permitted  to  remain  in  contact  with  the  clot  for 
three  hours.  If  it  is  desired  to  save  time,  the  clot,  as  soon  as 
formed,  may  be  cut  into  strips  and  placed  in  the  tubes  of  a 
centrifuge  and  whirled  for  a  half -hour.  This  secures  a 
greater  quantity  of  the  serum  and  at  the  same  time  gives  it 
its  full  value,  probably  by  injuring  the  leukocytes. 

Such  serum  containing  the  complement  is  useful  for  twenty- 


322    Wassermann  Reaction  for  Diagnosis  of  Syphilis 

four  hours.  Longer  it  should  not  be  kept  or  used,  as  it  begins 
to  deteriorate  almost  at  once,  and  the  deterioration  increases 
in  rapidity  in  proportion  to  the  length  of  time  it  is  kept.  The 
quantity  of  the  complement  in  the  serum  of  the  guinea-pig 
is  fairly  constant,  when  the  animal  is  regularly  fed,  and  fur- 
nishes a  fairly  uniform  reagent  that  requires  no  titration. 

II.  For  the  second,  or  hemolytic,  test  two  additional  re- 
agents are  required: 

Blood-corpuscles  to  be  dissolved. 

Hemolytic  amboceptors  by  which  complement  may  be 
united  to  them. 

(4)  The  Blood-corpuscles. — It  makes  no  difference  what 
kind  of  blood-corpuscles  are  employed.  Ehrlich  and  Mor- 
genroth,  in  their  pioneer  experiments  into  the  mechanism  of 
hemolysis,  used  goat  corpuscles.  Bordet  used  rabbit  cor- 
puscles; Wassermann,  Neisser,  and  Bruck,  sheep  corpuscles; 
Detre,  horse  corpuscles;  Noguchi,  human  corpuscles. 

As  those  who  do  many  tests  require  a  considerable  quantity 
of  blood,  it  seems  wisest  to  make  use  of  some  kind  that  is 
readily  obtainable  in  any  quantity,  hence  most  investigators 
now  follow  Wassermann  and  his  collaborators  and  use  sheep 
blood,  which  is  easily  obtained  at  a  slaughter-house  or  from 
sheep  kept  for  the  purpose. 

The  flowing  blood  is  caught  in  some  open  receptacle,  stirred 
until  it  is  defibrinated  (it  must  not  be  permitted  to  coagulate), 
and  then  taken  to  the  laboratory. 

The  corpuscles  must  next  be  washed  with  care,  so  as  to 
free  them  from  all  traces  of  amboceptors  and  complement  be- 
longing to  the  serum  in  which  they  are  contained.  For  this 
purpose  a  centrifuge  is  indispensable.  The  tubes  of  the 
apparatus  are  filled  with  the  defibrinated  blood  and  then 
whirled  for  fifteen  minutes  until  the  corpuscles  form  a  com- 
pact mass  below  a  fairly  clear  serum.  The  serum  is  then 
cautiously  removed  and  replaced  by  0.85  per  cent,  sodium 
chlorid  solution,  the  top  of  each  tube  closed  by  the  thumb,  and 
vigorously  shaken  so  as  to  distribute  the  corpuscles  through- 
out the  newly  added  fluid.  The  tubes  are  next  returned  to 
the  centrifuge  and  again  whirled  until  the  corpuscles  are 
sedimented,  when  the  fluid  resulting  from  this  first  wash- 
ing is  removed  and  replaced  by  fresh  salt  solution,  in  which 
the  corpuscles  are  again  thoroughly  shaken  up.  They  are 
now  again  whirled  until  again  sedimented,  when  the  second 
washing  is  removed,  leaving  the  corpuscular  mass  undis- 


The  Hemolytic  Amboceptor  323 

turbed.  Some  prefer  to  give  the  corpuscles  a  third  washing, 
but  it  does  not  seem  to  be  necessary.  Of  the  remaining 
corpuscular  mass,  5  c.c.  are  added  to  95  c.c.  of  salt  solution 
to  make  a  5  per  cent,  volume  suspension,  in  which  form  they 
are  ready  for  use.  As  the  corpuscles  of  healthy  sheep  thus 
treated  form  a  practically  invariable  unit,  no  titration  or 
other  preliminary  is  needed  before  they  are  used.  They 
must,  however,  be  used  within  twenty-four  hours  to  secure 
satisfactory  results,  as  they  tend  to  soften  when  kept  and  so 
to  lose  their  standard  value. 

(5)  The  Hemolytic  Amboceptor. — As  the  validity  of  the 
test  depends  upon  the  ability  or  inability  of  the  complement 
to  dissolve  the  corpuscles,  and  as  this  can  only  be  achieved 
when  appropriate  amboceptors  are  added,  the  hemolytic 
amboceptors  must  correspond  to  the  kind  of  blood-corpuscles 
employed  in  the  experiment.  As  has  been  shown,  the  greater 
number  of  investigators  now  employ  sheep  corpuscles,  hence 
must  use  such  corpuscles  as  the  antigen  through  whose  stimu- 
lation the  amboceptors  or  antibodies  are  excited. 

The  usual  method  of  obtaining  the  amboceptor  is  in  the 
blood-serum  of  an  experimentally  manipulated  rabbit.  A 
large  healthy  rabbit  is  employed  for  the  purpose,  and  is 
given  a  series  of  intraperitoneal  injections  of  the  5  per  cent, 
suspension  of  washed  and  sedimented  sheep  corpuscles  pre- 
pared as  above  described.  These  injections  are  usually  given 
about  five  days  apart,  and  the  dosage  is  usually  5,  10,  15,  20, 
and  25  c.c.  respectively. 

A  serum  of  higher  amboceptor  content  may  be  prepared  by 
using  a  greater  number  of  corpuscles,  and  for  this  purpose  the 
solid  corpuscular  mass  thrown  down  by  centrifugalization 
after  the  second  washing  is  employed.  Of  this,  2,  4,  8,  and 
12  c.c.,  diluted  with  just  enough  salt  solution  to  make  it 
pass  readily  through  the  hypodermic  needle,  may  be  regarded 
as  appropriate  doses,  the  intervals  being  the  same,  viz.,  five 
days.  The  amboceptor  content  of  the  rabbit  serum  seems  to 
be  greatest  about  the  ninth  or  tenth  day  after  the  last  injec- 
tion. Much  care  must  be  taken  to  see  that  the  injected  fluid 
is  sterile  and  the  operations  performed  under  aseptic  pre- 
cautions, as  the  rabbits  are  easily  infected  and  not  infre- 
quently die.  They  also  seem  prone  to  die  after  the  last  in- 
jection, so  that  it  is  best  to  have  more  than  one  rabbit  under 
treatment  at  a  time. 

When  the  appropriate  time  has  arrived  the  rabbit  is  bled 


324   Wassermann  Reaction  for  Diagnosis  of  Syphilis 

from  the  carotid  artery,  according  to  the  directions  given  in 
the  chapter  upon  Experiments  upon  Animals. 

The  blood  thus  obtained  is  permitted  to  coagulate,  and  the 
serum,  which  should  be  clear,  removed  with  a  pipet.  More 
serum  may  be  obtained  from  the  clot  by  cutting  it  into  strips, 
placing  these  in  a  centrifuge  tube,  and  whirling  them  for 
fifteen  minutes. 

Having  thus  described  the  preparation  of  the  reagents  to  be 
employed  in  making  the  Wassermann  test,  the  next  step,  that 
of  titrating  them,  becomes  essential.  One  of  the  first  ques- 
tions that  presents  itself  is  how  successful  titration  of  reagents 
that  may  all  be  more  or  less  variable  can  be  effected.  To 
achieve  this  it  is  necessary  to  begin  with  those  that  can  be 
assumed  to  present  the  least  variation  and  work  up  those  that 
are  most  variable. 

(1)  The  Sheep  Corpuscles. — As  these  come  from  a  healthy 
animal,  are  always  treated  in  precisely  the  same  manner  and 
used  under  standard  conditions  of  freshness,  they  can  be 
looked  upon  as  an  invariable  factor :  i  c.c.  of  the  5  per  cent, 
suspension  forms  a  good  working  quantity  and  constitutes 
I  unit. 

(2)  The  Normal  Guinea-pig  Serum  Containing  the  Comple- 
ment.— As  this  also  comes  from  a  normal  animal,  is  always 
treated  in  precisely  the  same  manner,  and  is  also  used  under 
standard  conditions  of  freshness,  etc.,  it  may  also  be  looked 
upon  as  a  factor  subject  to  very  slight  variation.     Of  this 
serum,  o.i  c.c.  (i  c.c.  of  a  i :  10  dilution)  forms  the  unit, 
or  working  quantity. 

These  two  reagents,  therefore,  may  be  regarded  as  the 
standards  of  measurement  through  which  the  titer  of  a 
third  is  made  possible. 

(3)  The  hemolytic  serum  from  the  rabbit  treated  with  the 
sheep  corpuscles. 

This  is  subject  to  very  great  variation,  according  to  the 
treatment  of  the  rabbit,  and  apparently,  also,  according  to 
the  ability  of  the  individual  rabbit  to  respond  to  the  treat- 
ment by  the  formation  of  hemolytic  amboceptors.  It  is, 
therefore,  imperative  to  make  a  careful  titration  of  this 
reagent. 

To  do  this  we  proceed  as  follows,  the  quantities  recom- 
mended being  such  as  experience  has  proved  most  satis- 
factory : 

Into  each  of  a  series  of  common  test-tubes  or  culture- 


The  Hemolytic  Amboceptor  -325 

tubes  i  c.c.  of  the  5  per  cent,  suspension  of  sheep  corpuscles 
and  i  c.c.  of  the  i :  10  dilution  of  the  normal  guinea-pig 
serum  (complement)  are  measured  with  graduated  pipets,  and 
then  to  each  of  these  tubes  the  rabbit  serum  (amboceptor) 
is  added  in  diminishing  quantities  for  the  purpose  of  de- 
termining the  least  quantity  that  will  bring  about  complete 
hemolysis  in  two  hours  at  the  temperature  of  37°  C.  The 
occurrence  of  the  hemolysis  is  shown  by  a  very  striking 
change  in  the  appearance  of  the  fluids.  The  mixture  is  at 
first  opaque  and  pale  red,  but  after  hemolysis,  or  solution 
of  the  red  corpuscles,  becomes  a  beautiful  transparent 
Burgundy  wine  red. 

The  actual  "  set-up  "  or  working  scheme  for  determining 
the  unit  or  least  hemolyzing  addition  of  the  amboceptor 
serum  may  be  represented  as  follows,  the  tubes  being  placed 
in  a  thermostat  and  observed  every  fifteen  minutes: 

Five  per  cent,  suspen-  Normal  guinea-pig  Hemolytic  rabbit  Result  (final  readings  after 
sion  of  corpuscles  (c.c.).  serum  (c.c.).  serum  (c.c.).  two  hours). 

o.oi  Complete  hemolysis. 

0.005 

0.002 

O.OOI 

0.0005 

0.0003  Partial  hemolysis. 
0.0002  No  hemolysis. 

o.oooi  " 


After  the  reagents  are  added,  enough  0.85  per  cent,  salt 
solution  is  added  to  each  tube  to  bring  the  total  bulk  of  the 
mixture  up  to  5  c.c. 

From  the  results  shown  in  the  tubes  it  is  evident  that  the 
hemolyzing  quantity  of  the  rabbit  serum  lies  between 
0.0005  and  0.0003  c.c.,  and  is  probably  0.0004  c-c-  To  be 
as  accurate  as  possible,  a  second  series  of  experiments  should 
be  made  with  0.0005,  0.00045,  and  0.0004  c-c->  so  that  the 
proportion  of  amboceptor  serum  necessary  to  effect  hemolysis 
be  known  within  small  limits.  This  least  quantity,  that  will 
certainly  cause  hemolysis  in  two  hours  at  37°  C.,  is  known 
as  the  unit.  The  combination  of  the  unit  of  corpuscular 
suspension  (i  c.c.),  the  unit  of  complement  (o.i  c.c.),  and  the 
unit  of  hemolytic  amboceptor  is  known  as  the  hemolytic 
system. 

As  soon  as  this  unit  is  known  accurately,  we  are  in  posi- 
tion to  reverse  the  conditions  of  the  test.  Thus,  if  we 
should  desire  to  know  how  much  variation  there  may  be  in 
the  complements  from  different  animals  under  different 


326    Wassermann  Reaction  for  Diagnosis  of  Syphilis 

conditions  of  age,  feeding,  health,  etc.,  we  can  now  do  so  by 
determining  whether,  when  i  c.c.  of  the  corpuscles,  i  unit 
of  amboceptor  and  varying  quantities  of  complementary 
serums  are  combined,  any  variation  in  the  final  results  will 
obtain. 

Or,  if  we  desire  to  know  to  what  extent  the  sheep  corpuscles 
may  change  through  prolonged  keeping  or  other  manipula- 
tion, it  can  be  done  by  maintaining  the  unit  of  amboceptor 
and  the  unit  of  complement  and  adding  larger  or  smaller 
quantities  of  the  corpuscles. 

The  conditions  under  which  the  unit  of  amboceptor  is 
titrated  constitute  the  standard  conditions  of  the  Wasser- 
mann reaction.  In  it  are  always  employed  i  unit  of  sheep 
corpuscle  suspension,  i  unit  of  complement,  and  i  unit  of 
amboceptor.  Here,  however,  a  slight  difference  of  opinion 
is  reached,  it  being  argued  by  many  experimenters  that  such 
exact  proportions  may  make  the  test  uncertain,  because, 
should  there  be  the  slightest  tendency  on  the  part  of  the 
remaining  reagents  to  inhibit  hemolysis  by  means  other 
than  complement  fixation,  it  would  result  in  positive  readings 
where  the  final  result  should  be  negative.  To  overcome  this 
possibility,  they  differentiate  between  the  amboceptor 
unit  and  the  amboceptor  dose,  the  latter  being  commonly 
twice  and  sometimes  four  times  the  unit. 

Now,  though  the  amboceptor  unit  is  determined  by  the 
method  given,  it  by  no  means  follows  that  those  proportions 
are  the  only  ones  that  will  lead  to  hemolysis.  By  increasing 
the  amboceptor  we  can  diminish  the  complement  with  the 
same  end-result,  a  matter  that  has  been  graphically  shown 
by  Noguchi,*  who  says  "  that  hemolysis  is  merely  the  rela- 
tive expression  of  the  combined  action  of  amboceptor  and 
complement,  and  is  not  the  absolute  indication  of  the  amount 
of  the  hemolytic  components  present  in  the  fluid.  The 
same  amount  of  hemolysis  can  be  produced  by  i  unit  of 
complement  and  by  i  unit  of  amboceptor  as  by  20  units 
of  amboceptor  and  o.i  unit  of  complement  or  any  other 
appropriate  combination  of  these  two  components." 

As  in  the  performance  of  the  test  we  work  always  with 
i  unit  of  complement,  we  do  not  want  to  unduly  disturb  its 
proper  proportional  action  by  any  excessive  addition  of  am- 
boceptor, but  simply  to  increase  the  latter  sufficiently  to  pro- 
vide for  the  accidental  presence,  in  the  serum  to  be  tested, 
*  "Serum  Diagnosis  and  Syphilis,"  1910,  p.  13  et  seq. 


The  Hemolytic  Amboceptor  327 

of  substances  affecting  hemolysis.  Fortunately,  means  are 
provided  for  controlling  this  action,  as  will  be  shown  below. 

The  amboceptor  serum  keeps  indefinitely.  When  it  is  to  be 
kept  and  used  from  time  to  time,  many  experimenters  prefer 
to  seal  it  in  a  number  of  small  tubes,  one  of  which  is  opened 
when  the  serum  is  needed,  the  remainder  being  kept  in  an 
ice-box.  Others  prefer  a  stoppered  bottle  that  can  be 
opened  and  a  measured  quantity  removed  as  needed.  The 
most  convenient  way  of  treating  it  seems  to  be  Noguchi's 
method  of  drying  it  upon  filter-paper. 

For  this  purpose  a  good  quality  of  filter-paper  is  cut  into 
strips  10  to  20  cm.  in  length  and  6  to  8  cm.  in  breadth,  and 
saturated  with  the  serum,  which  is  permitted  to  dry.  It  is 
well  to  make  a  preliminary  titration  of  the  serum,  for  if 
it  be  very  active  it  may  have  to  be  diluted  in  order  that  the 
piece  of  dry  paper  containing  the  dose  be  of  a  size  convenient 
to  handle;  i  drop  of  serum  usually  covers  about  J  sq.  cm., 
which  is  about  as  small  a  piece  as  can  be  measured,  cut,  and 
used  with  satisfaction  if  sufficient  allowances  are  to  be  made 
for  variations  in  distribution  and  other  conditions  that  may 
modify  the  accuracy  of  the  method.  If  the  unit-strength  of  a 
serum  be,  say,  0.00005  and  the  dose  o.oooi,  water  should  be 
added  to  the  extent  of  about  9  volumes  and  the  mixture 
gently  agitated,  so  that  diffusion  may  occur  without  frothing. 
The  diluted  serum  is  poured  into  a  large  flat  dish,  and  the 
strips  of  paper  passed  lengthwise  and  slowly  to  and  fro  until 
not  only  wet,  but  thoroughly  saturated.  Each  strip,  when 
the  dipping  is  finished,  is  held  first  by  one  end,  then  by  the 
other,  to  drain  off  the  free  drops,  and  then  laid  flat  upon  a 
clean  glass  plate  and  permitted  to  dry.  The  use  of  an  electric 
fan  is  recommended  to  hasten  drying.  Paper  so  prepared 
contains  everywhere  about  the  same  quantity  of  serum. 

The  real  titration  of  the  serum  now  begins.  With  a  ruler, 
one  piece  of  paper  is  divided  into  squares  of,  say,  J  cm., 
and  a  series  of  tubes  prepared  with  corpuscle  suspension  and 
complement  and  the  paper  added  i  square,  2  squares, 
2j  squares,  and  so  on  until  the  unit  is  determined.  When 
that  is  achieved,  the  exact  size  of  the  paper  containing  the 
unit  being  known,  one  sheet  of  the  paper  can  be  ruled  into 
squares  of  that  size  or  into  squares  of  twice  that  size — since 
the  "  dose  "  is  two  units — at  the  option  of  the  investigator. 

The  sheets  of  paper  are  kept  in  a  clean  envelope,  the  quan- 
tity for  each  test  being  cut  off  as  needed.  The  dry  serum 


328   Wassermann  Reaction  for  Diagnosis  of  Syphilis 

changes  so  little  that  the  dose  once  determined,  the  size  of  the 
square  of  paper  needed  for  the  test  remains  about  the  same. 

The  method  has  the  advantage  that  the  amboceptor  serum 
cannot  be  spoiled  or  spilled.  It  has  the  disadvantage  of 
being  slightly  less  accurate,  though  it  must  be  admitted  that 
the  chances  of  error  in  measuring  and  diluting  the  fluid 
serum  are  probably  as  great  as  those  arising  from  inequalities 
in  the  distribution  of  the  serum  throughout  the  paper. 

(4)  The  Antigen. — It  has  already  been  shown  that  com- 
plement is  labile,  and  it  may  have  occurred  to  the  reader 
that  its  activity  is  similar  to  that  of  ferments.  It  is  now 
necessary  to  point  out  the  many  conditions  (some  of  which 
may  arise  in  the  performance  of  a  test  so  delicate  as  the 
Wassermann  reaction)  by  which  the  complementary  action 
may  be  affected  or  set  aside.  Thus,  temperature  affects  it, 
and  temperatures  of  o°  C.  suspend  it.  It  is  on  this  account 
that  the  test  is  always  made  at  37°  C.  Like  most  of  the 
ferments  of  the  living  organism,  salts  affect  it,  and  in  salt- 
free  media  its  action  ceases,  to  return  when  a  small  quantity 
of  an  alkaline  salt  is  added.  Not  only  inorganic  salts,  but 
salts  of  the  fatty  acids  and  the  bile-salts  may  inhibit  it. 
Certain  lipoids,  such  as  lecithin,  cholesterin,  protogon  and 
tristearin,  and  neutral  fats  inhibit  the  complementary  action. 
Some  of  these  substances  are  always  present  in  the  serum 
containing  the  complement  itself  or  in  the  other  serums  to 
be  tested  by  its  use,  and,  as  Wassermann  and  Citron  have 
pointed  out,  we  really  know  nothing  about  complementary 
action.  Aleuronat,  inulin,  peptone,  albumose,  tuberculin, 
natural  and  artificial  aggressins,  gelatin,  casein,  sitosterin, 
coagulated  serum-albumin,  and  albuminous  precipitates  all 
act  as  inhibitives  to  complementary  action. 

Now,  in  all  combinations  of  several  serums  and  antigens  it 
is  always  possible  that  some  of  these  complement-binding  or 
complement-inhibiting  substances  may  be  present,  hence  the 
first  thing  that  has  to  be  done  in  the  way  of  titrating  the 
antigen — which  is  a  tissue  extract,  rich  in  lipoids  which  in- 
hibit complementary  action — is  to  determine  how  much  of  it 
can  be  added  to  the  "  hemolytic  system  "  without  disturbing 
hemolysis. 

As,  however,  the  antigen  is  not  used  by  itself,  but  always 
in  combination  with  a  serum  to  be  tested,  we  must  always 
combine  it  with  serum  when  making  the  titration,  so  that  the 
requirements  of  the  test  may  be  conformed  with.  In  order 


The  Hemolytic  Amboceptor  329 

that  the  essential  difference  between  the  normal  serum  and 
the  syphilitic  serum  can  be  reduced  to  precise  calculation  it 
is  imperative  that,  in  all  the  tests,  the  same  quantity  of 
added  serum  be  employed.  Experience  has  shown  this 
quantity  to  be  0.2  c.c.,  and  this  we  regard  as  the  unit  of 
serum  to  be  tested. 

To  titrate  the  antigen  we  require  (i)  a  normal  human 
serum  and  (2)  a  known  syphilitic  serum,  obtained  from 
blood  drawn  from  the  arm  veins  of  cases  known  to  be  well 
and  cases  known  to  be  syphilitic  respectively.  These  serums 
should  be  kept  on  hand  in  the  laboratory  in  consider- 
able quantity,  as  they  are  constantly  needed  for  making  the 
controls  that  must  accompany  each  test,  as  well  as  for  making 
the  preliminary  titration  of  the  antigen. 

The  "  set-up  "  for  the  titration  of  antigen  is  fairly  simple. 
A  series  of  tubes  is  prepared  and  divided  into  two  groups. 
Into  each  tube  in  each  group  is  placed  i  unit  of  complement. 
Each  tube  of  one  group  receives  the  addition  of  0.2  c.c.  of  the 
normal  serum;  each  tube  of  the  other  group,  0.2  c.c.  of  the 
known  syphilitic  serum.  All  the  tubes  now  receive  additions 
of  antigen,  so  that  one  tube  of  each  group  contains  the  same 
quantity.  The  quantity  of  antigen  not  being  known,  it  is 
only  through  the  experience  of  others  that  we  can  guess  where 
to  start.  An  idea  can  be  formed  through  study  of  the  follow- 
ing tabulation: 

TABLE  I.  —  Series  "with  the  Normal  Serum, 
Tubes. 

1.  i  unit  of  +       i  unit  of       -f-  antigen  o.oi  u  &^  S      =  Complete 
complement  normal  serum                               °«--5.c  ^          hemolysis. 

2.  i  unit  of  -|-       i  unit  of       +  antigen  0.03  *»«ga  5      =  Complete 
complement  normal  serum                                5^    •*          hemolysis. 

3.  i  unit  of  +       i  unit  of       +  antigen  0.05  *1  §J  %      =  Complete 
complement  normal  serum                                |-^-^          hemolysis. 

4.  i  unit  of  +       i  unit  of       +  antigen  0.07  jg.«  £  i      =  Complete 
complement  normal  serum                               t^  1  o          hemolysis. 

5.  i  unit  of  +       i  unit  of       +  antigen  0.08  £  g  ^~_  =  Complete 
complement  normal  serum                               •-w'S.tnlJ       hemolysis. 

6.  i  unit  of  +       i  unit  of       +  antigen  0.09  "?•§  §  1-|  =  Complete 
complement  normal  serum                               !~-3  jjj       hemolysis. 

7.  i  unit  of  +       i  unit  of       +  antigen  o.i     B*i  *         =  Complete 
complement  normal  serum                                 t<u<o^rt       hemolysis. 

8.  i  unit  of  +       i  unit  of       +  antigen  0.12  J  -3  03  ft—  Complete 
complement        normal  serum  'g  g-cTg^g       hemolysis. 

9.  i  unit  of  +       i  unit  of       +  antigen  0.15  «•£  M  g  g  =  Complete 
complement        normal  serum  |C  ^  1  2  «,       hemolysis. 

10.     i  unit  of      +       i  unit  of       +  antigen  0.18  g.1  f  f*l  —  Complete 
complement        normal  serum  £  „•§  S^       hemolysis. 

i  unit  of     -{-        i  unit  of       +  antigen  0.2    bog.o^=No 
complement        normal  serum  jjj  =3-5       hemolysis. 


ii 


330   Wassermann  Reaction  for  Diagnosis  of  Syphilis 

TABLE  II. — Series  with  the  Syphilitic  Serum. 
Tubes. 

1.  i  unit  of     +       i  unit  of       •+-  antigen  o.oi  «'2  jjjj      =  Complete 
complement      syphilitic  serum  «^°          hemolysis. 

2.  i  unit  of     +       i  unit  of       +  antigen  0.03  g^  G~§     =  Complete 
complement      syphilitic  serum  §-o|^^       hemolysis. 

3.  i  unit  of      +        i  unit  of       +  antigen  0.05  g%  >- ^'5  —  Sugges- 
complement      syphilitic  serum  sSftSj          tion  of 

**  <u  g  §S       hemolysis. 

4.  i  unit  of     +       i  unit  of       +  antigen  0.07  .S^-2  •£  °  =  Slight 
complement      syphilitic  serum  ^M  *£ «       hemolysis. 

6.     i  unit  of     +       i  unit  of       +  antigen  0.08  |  g"3  £  |  =  Partial 
complement      syphilitic  serum  "o^^^       hemolysis. 

6.  i  unit  of      +       i  unit  of       +  antigen  0.09  H'^'S  g  a  =  No 
complement      syphilitic  serum  ^- ^  H  a  «       hemolysis. 

7.  i  unit  of     +       i  unit  of       +  antigen  o.i    .*^|  S'g  =  No 
complement      syphilitic  serum  2  §  <g  H^       hemolysis. 

8.  i  unit  of     +       i  unit  of       +  antigen  0.12  ^Irf  gt>  :=^No 
complement      syphilitic  serum  J>  g  §.•§£       hemolysis. 

9.  i  unit  of     +       i  unit  of       +  antigen  0.15  „  g  8  «  g  =  No 
complement      syphilitic  serum  Jt  g-"5"!       hemolysis. 

10.     i  unit  of     +       i  unit  of       +  antigen  0.18  b^-Siy  £  =  No 

complement      syphilitic  serum  °ST13'I  j|       hemolysis. 


From  this  we  find  that  the  unit  of  antigen  is  0.09  c.c. 
The  largest  quantity  of  the  antigen  that  can  be  added 
without  preventing  hemolysis  when  the  normal  serum  is 
used  is  probably  0.18  c.c.  At  the  same  time  0.09  c.c.  is 
the  smallest  quantity  that  can  be  added,  when  the  syph- 
ilitic serum  is  used,  to  prevent  it.  In  this  case  the  dose 
exactly  fulfils  Kaplan's  requirement  that  "  The  unit  dose  of 
antigen  must  completely  inhibit  hemolysis  ...  of  a  known 
luetic  serum,  provided  double  the  dose  does  not  interfere 
with  the  complete  hemolysis  of  cells  using  a  known  normal 
serum  and  complement." 

We  have  now  accomplished  the  titration  of  all  five  of  the 
factors  involved  in  making  the  Wassermann  reaction,  but 
we  have  done  more,  we  have  really  done  the  test,  and  have 
seen  positive  and  negative  results,  for  in  titrating  the  anti- 
gen we  have  developed  the  reaction  by  which  we  can  confirm 
the  diagnosis  of  syphilis  in  the  case  from  whom  the  syphilitic 
serum  was  obtained,  and  have  failed  to  develop  it  with  the 
known  normal  serum. 

However,  in  order  that  those  who  perform  the  test  may  be 
able  to  escape  the  numerous  errors  into  which  one  may  fall, 
it  will  be  necessary  to  point  out  the  controls  by  which  they 
can  be  escaped. 

A  Wassermann  reaction  at  the  present  time  comprises  not 
only  the  test  of  the  patient's  serum,  but  simultaneously  in- 
cludes a  long  series  of  other  tests  by  which  the  validity  of 


"S.3 
i£.M 

fll 


TEST. 

Tube  containing  the  serum  to  be  tested. 


1 

II 

C*  f- 
•R? 

h* 

CONTROL. 
Control  of  serum  to  be 


ested   to  deter- 


mine    substances    which    without    antigen  „ 
may  inhibit  hemolysis.  g. 


R          §> 

|          || 

* 

f       ^r 

IJS, 

girt 


^  CONTROL. 

<  "2.  |^  «      Control  of  the  test  by  the  use  of  a  known 
~     S'  '    syphilitic  serum. 


CONTROL. 

Control  of  the  known  positive  to  deter- 
mine that  it  contains  no  recently  developed 
. «.  -T*1  substances  that  may  inhibit  hemolysis. 


i  i 

- 

CONTROL. 

«•      Control  of  the  test  by  the  use  of  a  known 
norma   serum. 


\  K 

L  ^^ 

pi 

1  oddf-oJc-c. 

. 

- 

a  £• 


CONTROL. 

3  Control  of  the  known  normal  serum  to  de- 
3  termine  that  no  substances  inhibiting  hemo- 
H  lysis  had  developed  in  it. 

I 


a  »: 


the  first  part  of 
n  the  thermosta 


1>  ^ 

2t 

n 


CONTROL. 

Control  test  to  determine  changes  in  the 
?T  antigen  by  which  hemolysis  might  be  pre- 
^"  vented. 


!? 

5s 

l| 

R|' 

- 

CONTROL. 
Control  of  the  hemolytic  system. 


* 


CONTROL.  n 

Control  for  the   purpose  of  determining  • 
the  presence  of  anti-sheep  amboceptors   in 
the  serum  to  be  tested. 


331 


1    tt 

I    % 

<to 

1?       2 

332    Wassermann  Reaction  for  Diagnosis  of  Syphilis 

every  part  of  the  test  and  the  correct  titer  of  all  the  reagents 
employed  can  be  simultaneously  ascertained.  Every  one 
who  makes  the  test  should  practice  some  such  systematic 
method  as  is  suggested  by  the  following  scheme  for  the  "  set- 
up." Nine  tubes  are  employed  for  the  usual  test.  These  are 
stood  in  a  rack  in  the  same  order  for  every  test,  and  in  the 
course  of  time  it  becomes  a  matter  of  habit  to  know  the  tubes 
by  number,  and  to  recall  for  what  each  stands. 

If  many  tests  are  to  be  made  at  one  time,  it  is,  of  course, 
unnecessary  to  make  more  than  one  series  of  the  various  con- 
trols. 

Of  the  complementary  serum  we  add  i  c.c.  to  9  c.c.  of  0.85 
per  cent,  (physiologic)  salt  solution,  making  each  cubic  centi- 
meter of  the  dilution  of  the  fluid  equal  o.i  c.c.  This  quan- 
tity, carefully  measured  by  the  same  volumetric  pipet,  is 
dropped  into  each  tube,  and  this  pipet  laid  aside. 

The  serum  to  be  tested  is  drawn  into  a  second  finely  gradu- 
ated pipet,  and  0.2  c.c.  added  to  tubes  i,  2,  and  9,  and  that 
pipet  laid  aside. 

The  positive  syphilitic  serum  used  to  control  the  test  is 
similarly  drawn  up  in  a  fresh  pipet  and  0.2  c.c.  of  it  measured 
into  tubes  3  and  4,  and  the  pipet  laid  aside. 

The  normal  serum  used  as  a  control  is  similarly  drawn  into 
still  another  pipet  and  0.2  c.c.  measured  into  tubes  5  and  6, 
and  the  pipet  laid  aside. 

The  alcoholic  extract  composing  the  antigen  is  next  added, 
either  by  diluting  it  so  that  i  c.c.  contains  the  unit,  or  measur- 
ing the  unit  quantity  directly  into  the  tubes.  The  antigen  is 
added  to  tubes  i,  3,  5,  and  7,  and  the  pipet  laid  aside. 

Lastly,  each  tube  receives  a  correctly  measured  quantity 
of  0.85  per  cent,  sodium  chlorid  solution  to  bring  the  total 
bulk  of  fluid  up  to  exactly  3  c.c. 

Each  tube  is  now  shaken  carefully,  so  as  not  to  cause  froth- 
ing of  the  fluid,  and  the  rack  is  stood  in  a  thermostat  kept 
at  37°  C. 

At  the  end  of  an  hour  the  rack  is  removed,  and  every  tube 
receives  the  addition  of  i  unit  of  the  sheep  corpuscle  sus- 
pension and,  with  the  exception  of  tube  9,  receives  one  dose 
of  amboceptor,  either  the  serum  measured  by  diluting  so  that 
i  c.c.  equals  the  dose,  or  the  necessary  square  of  paper.  This, 
in  the  former  case,  brings  the  total  bulk  of  fluid  to  5  c.c.,  in 
the  latter  makes  it  necessary  to  add  i  more  cubic  centimeter 
of  salt  solution  to  each  tube.  We  aim  to  have  exactly  5  c.c. 
of  fluid  in  each  tube. 


The  Hemolytic  Amboceptor 


333 


The  tubes  are  again  stood  in  the  thermostat,  where  they 
are  permitted  to  remain  for  an  hour,  when  the  readings  are 
taken  and  carefully  noted.  After  this  the  rack  and  all  the 
tubes  are  placed  in  the  ice-box  until  twenty-four  hours'  old, 
when  the  final  readings  are  taken  and  the  conclusions  are 
reached. 

As  a  rule,  the  readings  taken  after  the  second  hour  of  in- 
cubation and  those  taken  after  twenty-four  hours  correspond. 

A  valid  test  should  show  the  following: 


Test  Controls. 


Tubes. 
I. 

2. 

3- 
4- 

6. 

8. 
9- 


No  hemolysis  in  syphilis. 
Complete  hemolysis. 


Hemolysis  in  health. 


No  hemolysis  (this  is  the  standard  of  comparison). 
Complete  hemolysis. 


No  hemolysis,  as  a  rule. 


Fig.  101. — A  typical  positive  Wassermann  reaction  with  the  recom- 
mended controls  as  it  appears  after  standing  twelve  hours.  Corpus- 
cular sedimentation  without  hemolysis  is  seen  in  tubes  i,  3,  and  9; 
complete  hemolysis  in  the  others. 


In  the  tubes  in  which  hemolysis  takes  place  the  change  is 
very  marked.     The  hemoglobin  dissolves  out  of  the  corpus- 


334   Wassermann  Reaction  for  Diagnosis  of  Syphilis 

cular  stroma  and  saturates  the  fluid,  transforming  it  from 
the  opaque  pale  red  to  a  transparent  Burgundy  red.  Some- 
times the  corpuscular  stroma  dissolves,  sometimes  it  sedi- 
ments as  a  colorless  mass  to  the  bottom  of  the  tube. 

In  the  tubes  containing  the  positive  or  syphilitic  serum,  and 
in  which  there  is  complete  complement  fixation,  the  unaltered 
corpuscles  sediment  to  the  bottom  of  the  tube,  leaving  a 
colorless  fluid  above. 

When  the  complement  fixation  is  complete  there  is  no  so- 
lution of  the  hemoglobin.  Such  a  result  has  been  described 
by  Citron  as  H — \-  +  + .  When  the  sedimented  corpuscles  lie 
at  the  bottom  of  a  slightly  reddened  fluid,  the  result  is  said  to 
be  +  +  +  ;  when  at  the  bottom  of  a  distinctly  red  fluid,  +  + , 
etc.  Confusion  will  be  avoided  by  making  reports  as  positive 
in  all  cases  in  which  there  is  a  distinct  red  corpuscular  de- 
posit, regardless  of  the  state  of  the  supernatant  fluid,  and 
negative  when  there  is  no  such  deposit. 

When  we  come  to  inquire  why  the  supernatant  fluid  should 
be  red,  we  reach  a  question  that  is  not  quickly  answered. 
In  order  to  be  in  a  position  to  explain  it  in  certain  cases  we 
introduced  in  our  series  tube  9,  by  which  to  discover  whether 
the  serum  under  examination  contains,  as  is  sometimes  the 
case  in  health  as  well  as  in  syphilis,  rabbit  corpuscle  ambo- 
ceptors.  If  tube  9  shows  such  amboceptors  to  be  in  the 
serum,  it  explains  the  redness  of  the  fluid  bathing  the  cor- 
puscles, and  does  not  invalidate  the  test.  If  no  such  ambo- 
ceptors are  present  and  the  fluid  is  still  red,  it  may  indicate 
that  a  little  of  the  complement  remained  unfixed  and  acted 
upon  a  few  of  the  corpuscles. 

The  Validity  of  the  Test. — The  Wassermann  reaction  is  not 
a  certain  test  for  syphilis.  It  is  an  aid  in  making  the  diag- 
nosis, especially  in  cases  in  which  there  are  no  symptoms. 

Of  thousands  of  bloods  of  normal  persons  examined,  the 
results  are  almost  100  per  cent,  negative.  Basset-Smith  has 
had  a  positive  reaction  in  a  case  of  scarlet  fever  and  one  in  a 
case  of  malignant  disease  of  the  liver  with  jaundice ;  Oppen- 
heim,  one  in  a  case  of  tumor  of  the  cerebellopontine  angle; 
Marburg,  one  in  a  similar  case;  Newmark  reports  2  cases  of 
brain  tumors  with  positive  reactions;  Cohn,  a  positive  in  a 
patient  with  a  cerebral  tumor.  These  seem  to  form  the  greater 
part  of  positive  reactions  in  non-syphilitics  thus  far  recorded. 

In  active  syphilis  Wassermann  had  90  per  cent,  of  positive 
reactions  in  2990  cases;  and  most  others  report  about  the 


Noguchi 's  Modification  335 

same.  Basset-Smith  in  458  such  cases  found  94  per  cent, 
positive  reactions. 

In  latent  syphilis  Wassermann  found  50  per  cent,  positive 
reactions;  Basset-Smith,  46  per  cent. 

In  chronic,  presumably  syphilitic,  disease  of  the  nervous 
system,  general  paresis,  and  tabes  dorsalis  the  positive  reac- 
tions vary.  In  the  former  disease  some  have  found  as  high 
as  90  per  cent,  positive;  in  the  latter  the  usual  figures  vary 
about  50  per  cent. 

It  is  thus  seen  that  the  occurrence  of  the  reaction  is  much 
more  conclusive  evidence  of  the  presence  of  syphilitic  infection 
than  the  failure  of  the  reaction  is  of  its  absence. 

Treatment  greatly  influences  the  test.  When  under  active 
treatment,  either  with  mercury  and  iodids  or  with  salvarsan, 
the  reaction  of  the  serums  is  usually  negative. 

Nature  of  the  Reaction. — We  now  reach  the  point  of  con- 
sidering the  nature  of  the  reaction.  It  is  certainly  not  a  vari- 
ation of  the  Bordet-Gengou  phenomenon.  It  does  not  occur 
because  of  the  presence  in  the  blood  of  syphilitics  of  antibodies 
which  combine  with  the  antigen  and  fix  the  complement. 
It  is  probably  not  complement  fixation  so  much  as  comple- 
mentary inhibition,  through  the  presence  in  the  blood  of 
syphilitics  of  certain  metabolic  products,  whose  action  inter- 
feres with  the  complement  in  some  entirely  different  manner. 

NOGUCHFS    MODIFICATION    OF    THE    WASSERMAMN 
REACTION. 

Noguchi*  has  modified  the  Wassermann  reaction,  first  by 
employing  as  an  antigen  an  extract  of  the  heart  of  a  normal 
guinea-pig,  and,  second,  by  making  use  of  human  instead  of 
sheep  corpuscles  for  the  hemolytic  test.  The  advantage 
of  the  latter  depends  upon  the  fact,  carefully  determined  by 
Noguchi,  that  human  blood-serum  contains  no  amboceptors 
active  in  effecting  hemolysis  of  human  blood-corpuscles, 
though  it  not  infrequently  contains  hemolytic  amboceptors 
for  sheep  corpuscles.  In  the  directions  for  making  the  Was- 
sermann test  a  control  test  for  determining  their  presence  or 
absence  was  found  expedient.  It  will  also  be  remembered 
that  the  presence  of  these  amboceptors  causes  no  invalidity 
of  the  test,  provided  it  be  recognized. 

*  "Serum  Diagnosis  of  Syphilis,"  Philadelphia,  1910,  J.  B.  Lippin- 
cott  Co. 


33 6   Wassermann  Reaction  for  Diagnosis  of  Syphilis 

Noguchi  also  varies  the  technic  in  such  a  manner  that  very 
small  quantities  of  the  various  reagents  are  employed — a 
necessity  that  arises  from  the  relatively  small  quantity  of  the 
patient's  blood  obtainable  according  to  the  method  he  em- 
ploys. The  reagents  employed  are  as  follows: 

(1)  The  Serum  to  be  Tested. — To  obtain  this,  Noguchi  binds 
the  finger  of  the  patient  with  a  rubber  band,  makes  a  good- 
sized  puncture  near  the  root  of  the  nail  with  a  Hagedorn 
needle,  and  collects  about  2  c.c.  of  the  blood  in  a  Wright  tube 
(see  directions  for  making  the  opsonic  index).      The  blood 
soon  coagulates  in  the  tube,  which  is  then  scratched  with 
a  diamond  or  file,  broken,  and  the  serum  removed  with  a 
capillary  pipet.     The  serum  may  or  may  not  be  inactivated 
by  heat,  according  to  the  option  of  the  experimenter.     The 
dose  of  the  unheated  serum  is  i   drop;  of  the  inactivated 
serum,  4  drops.     The  same  doses  of  the  normal  and  syphilitic 
control  serums  are  used. 

(2)  The  Complement. — This  consists  of  fresh  guinea-pig 
serum.     Of  it  he  makes  a  40  per  cent,  dilution  in  physiologic 
salt  solution  by  adding  one  part  of  the  serum  to  i^  parts  of 
the  salt  solution;  o.i  c.c.  is  the  unit.     Two  units  constitute 
the  "  dose." 

(3)  The  Antigen. — The  antigen  is  made,  according  to  the 
directions   given    in    the    description   of   the    Wassermann 
test,  out  of  normal  guinea-pig  heart.     The  extract  is  dried 
upon  filter-paper,  as  has  been  recommended  for  the  hemolytic 
amboceptor,  and  titrated  according  to  the  size  of  the  square 
of  paper  needed,  instead  of  the  quantity  of  fluid  to  be  added. 

(4)  The  Corpuscle  Suspension. — For  this  purpose  either 
normal  human  corpuscles  or  the  corpuscles  of  the  patient 
whose  blood  is  to  be  examined  may  be  employed.    Instead  of 
a  5  per  cent,  suspension  a  i  per  cent,  suspension  is  recom- 
mended.   If  normal  corpuscles  are  employed,  it  is  necessary  to 
wash  them  free  of  the  normal  serum  or  plasma,  which  Noguchi 
accomplishes  as  follows:  8  c.c.  of  normal  salt  solution  are 
placed  in  a  large  test-tube,  and  the  blood  flowing  from  a  punc- 
ture (in  the  operator's  own  finger,  for  example)  permitted  to 
drop  in,  the  proportion  being  i  drop  to  each  4  c.c.     The  fluid 
is  then  shaken  and  stood  on  ice  over  night,  when  the  corpuscle 
sediment  and  the  supernatant  fluid  containing  the  fibrin 
factors  and  ferment  is  decanted  and  replaced  by  fresh  salt 
solution,  and  the  suspension  made  by  shaking.     Or,  in  a 
laboratory,  the  corpuscles  can  be  washed  as  usual  with  the 


Noguchi's  Modification 


337 


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Incubation  at  37°  C.  for  2  hours  longer, 
then  at  room  temperature. 


I! 


338    Wassermann  Reaction  for  Diagnosis  of  Syphilis 

aid  of  the  centrifuge.  If  the  patient's  own  corpuscles  are  to 
be  employed,  some  of  them  may  be  distributed,  without  any 
washing,  through  the  serum  by  simply  shaking  it  up  a  little 
with  the  clot.  It  is  not  essential  exactly  to  measure  the 
corpuscles,  as  after  a  few  trials  with  the  suspension  of  normal 
corpuscles  the  eye  becomes  accustomed  to  the  color,  intensity, 
and  density  corresponding  to  the  requirement. 

(5)  The  Antihuman  Hemolytic  Amboceptor. — This  is  pre- 
pared by  injecting  rabbits,  according  to  the  method  already 
described,  with  washed  human  corpuscles  obtained  from  fresh 
human  placentae  or  from  the  heart  of  a  fresh  cadaver  come  to 
autopsy.  The  serum  of  the  rabbit,  when  obtained,  is  dried 
upon  blotting-paper  and  titrated  as  already  described. 

The  "  set-up  "  for  the  test,  as  given  by  Noguchi,  is  less 
cumbersome  than  that  recommended  for  the  Wassermann 
test  and  includes  six  tubes.  It  can  best  be  understood  by 
reference  to  the  diagram  on  page  337. 

The  method  recommends  itself  through  its  simplicity  and 
convenience,  no  sheep  corpuscles  being  used,  and  through 
the  smaller  quantity  of  blood  required,  it  seeming  to  the 
patient  that  less  damage  is  done  by  pricking  the  finger  than 
by  introducing  a  syringe  needle  into  a  vein.  It  is,  moreover, 
a  very  sensitive  test,  and  gives  very  accurate  results  as  far 
as  regards  positive  cases.  Unfortunately,  it  seems  to  have 
the  demerit  of  occasionally  finding  the  reaction  in  negative 
cases,  which  is  a  serious  defect. 

Diagnosticians  are  still  divided  in  opinion,  some  preferring 
the  Wassermann  test,  some  the  Noguchi  test,  and  some  always 
doing  both,  permitting  the  one  to  control  the  other.  In  the 
long  run  the  Wassermann  test  seems  to  meet  with  most  favor, 
and  in  the  hands  of  the  majority  leads  to  most  satisfactory 
results. 


PART   II. 

THE  INFECTIOUS  DISEASES  AND  THE 
SPECIFIC   MICRO-ORGANISMS. 


CHAPTER   I. 

SUPPURATION. 

SUPPURATION  was  at  one  time  looked  upon  as  a  normal 
and  inevitable  outcome  of  the  majority  of  wounds,  and 
although  bacteria  were  early  observed  in  the  purulent  dis- 
charges, the  insufficiency  of  information  then  at  hand  led  to 
the  belief  that  they  were  spontaneously  developed  there. 

From  what  has  already  been  said  about  the  evolution  of 
bacteriology  and  the  biology  and  distribution  of  bacteria, 
the  relationship  existing  between  bacteria  and  suppuration, 
and,  indeed,  between  bacteria  and  disease  in  general,  is 
found  to  be  reversed.  Instead  of  being  the  products  of 
disease,  we  now  know  that  the  micro-organisms  are  the 
cause 

With  this  altered  point  of  view  came  the  question,  Whence 
come  the  micro-organisms  that  cause  disease?  The  wide 
distribution  of  bacteria  in  the  air  naturally  led  surgeons  to 
look  upon  it  as  the  source  of  all  infection,  and  to  make  most 
strenuous,  though  mistaken  efforts  to  disinfect  it,  that  it 
might  not  contaminate  wounds. 

The  development  of  antiseptic  surgery,  and  the  extremes 
to  which  the  application  of  germicides  was  carried,  became 
almost  ridiculous,  for  not  only  were  the  hands  of  the  opera- 
tor, his  instruments,  sponges,  sutures,  ligatures,  and  dress- 
ings kept  constantly  saturated  with  powerful  and  irritating 
germicidal  solutions,  and  the  wound  subsequently  covered 
by  dressings  saturated  with  germicides,  but  by  means  of 

339 


340  Suppuration 

a  steam  atomizer  the  air  over  the  wound  was  kept  filled 
with  a  disinfecting  vapor  during  the  whole  operation. 

More  recent  researches,  however,  have  shown  not  only 
that  the  atmosphere  need  not  be  disinfected,  but  also  that  the 
air  of  ordinarily  quiet  rooms,  while  containing  a  few  sapro- 
phytic  organisms,  very  rarely  contains  pathogenic  bacteria, 
and  is  rarely  an  important  factor  in  wound  infection.  A 
direct  stream  of  air,  such  as  is  generated  by  an  atomizer, 
really  directs  more  bacteria  toward  the  wound  than  would 
ordinarily  fall  upon  it,  thereby  increasing,  instead  of  lessen- 
ing, the  danger  of  infection. 

The  strong  disinfecting  solutions  once  employed  have  like- 
wise been  largely  abandoned,  the  modern  view  being  that  it 
is  far  wiser  to  prevent  the  entrance  of  organisms  into  wounds 
than  to  destroy  them  by  the  application  of  strong  and 
irritating  solutions. 

Suppuration,  while  nearly  always  the  result  of  micro- 
organismal  activity,  is  not  a  specific  infectious  process,  but 
the  expression  of  a  violent  tissue-reaction  that  may  result 
from  various  injurious  agents. 

Being,  therefore,  but  the  expression  of  tissue  irritation 
arising  through  strong  chemotactic  influences,  it  is  only  to 
be  expected  that  as  many  bacteria  may  be  associated  with 
it  as  can  bring  about  the  essential  conditions.  Bacteria  with 
which  these  qualities  are  exceptionally  marked  appear  as  the 
common  cause  of  the  process;  those  with  which  it  is  less 
marked,  as  exceptional  causes. 

Attention  has  already  been  called  to  the  fact  that  certain 
micro-organisms  are  so  intimate  in  their  relation  to  the  skin 
that  it  is  almost  impossible  to  get  rid  of  them,  and  in  this  rela- 
tion the  experiments  of  Welsh,  Robb,  and  Ghriskey  upon 
hand  disinfection  have  been  cited.  These  observers  have 
shown  that,  no  matter  how  rigid  the  disinfection  of  the 
patient's  skin,  the  cleansing  of  the  operator's  hands,  the 
sterilization  of  the  instruments,  and  the  precautions  exer- 
cised, a  certain  number  of  wounds  in  which  sutures  are  em- 
ployed will  always  suppurate,  the  cause  of  the  suppuration 
being  the  skin  cocci,  all  of  which  it  is  impossible  to  remove. 
We  thus  carry  infectious  organisms  constantly  with  us  upon 
our  skins,  and  so  pave  the  way  for  suppuration  in  wounds. 
That  all  wounds  do  not  suppurate  probably  depends  largely 
upon  the  local  and  general  immunity  of  the  individual, 
rather  than  upon  the  absence  of  organisms  from  the  wounds. 


Staphylococcus  Epidermic! is  Albus  341 

The  relative  frequency  with  which  certain  varieties  of 
bacteria  are  associated  with  suppuration  is  well  shown  in 
the  following  table  from  Karlinski:* 

Suppuration  in  man —                        Streptococci,  45  cases. 

Staphylococci,  144 

Other  bacteria,  15 

Suppuration  in  the  lower  animals — Streptococci,  23 

Staphylococci,  45 

Other  bacteria,  15 

Suppuration  in  birds —                        Streptococci,  1 1 

Staphylococci,  40 

Other  bacteria,  20 

Andrewes  and  Gordon,  f  after  the  examination  of  large 
numbers  of  Staphylococci  from  lesions  of  the  human  skin 
and  mucous  membranes,  came  to  the  conclusion  that  four 
varieties  are  differentiable.  Of  these,  the  Staphylococcus 
pyogenes  is  the  most  common  and  most  important.  When 
typical,  this  produces  an  orange-colored  pigment.  When 
atypical,  it  may  be  lemon  yellow  or  white.  Staphylococcus 
epidermidis  albus  is  a  distinct  species.  The  differences 
between  these  cocci  are  shown  in  the  table  on  page  342. 


STAPHYLOCOCCUS  EPIDERMIDIS  ALBUS  (WELCH). 

General  Characteristics. — A  non-motile,  non-flagellate,  non- 
sporogenous,  slowly  liquefying,  non-chromogenic,  aerobic  and  optionally 
anaerobic,  doubtfully  pathogenic  coccus,  staining  by  the  usual  methods 
and  by  Gram's  method,  and  having  its  natural  habitat  upon  the  skin. 

Under  the  name  Staphylococcus  epidermidis  albus,  Welch  J 
has  described  a  micrococcus  which  seems  to  be  habitually 
present  upon  the  skin,  not  only  upon  the  surface,  but  also 
deep  down  in  the  Malpighian  layer.  He  believes  it  to  be 
Staphylococcus  pyogenes  albus  in  an  attenuated  condition, 
and  if  this  opinion  be  correct,  and  there  is  seated  deeply 
in  the  derm  a  coccus  which  may  at  times  cause  suppuration, 
the  conclusions  of  Robb  and  Ghirskey,  that  sutures  of  cat- 
gut when  tightly  drawn  may  be  a  cause  of  skin-abscesses  by 
predisposing  to  the  development  of  this  organism,  are  cer- 
tainly justifiable.  As  the  morphologic  and  cultural  char- 
acteristics of  the  organism  correspond  fairly  well  to  those  of 

*  "Centralbl.  f.  Bakt.,"  etc.,  vn,  S.  113,  1890. 

t  "Report  of  the  Local  Government  Board  of  Great  Britain,"  Sup- 
plement; "Report  of  the  Medical  Officers,"  1905-06,  vol.  xxxv,  p.  543. 
|  "Amer.  Jour.  Med.  Sci.,"  1891,  p.  439. 


342 


Suppuration 


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Staphylococcus  Pyogenes  Aureus  343 

the  following  species,  no  separate  description  of  them  seems 
necessary. 

STAPHYLOCOCCUS  PYOGENES  ALB  us  (ROSENBACH*). 

General  Characteristics.— A  non-motile,  non-flagellate,  non- 
sporogenous,  liquefying,  non-chromogenic,  aerobic  and  optionally 
anaerobic,  mildly  pathogenic  coccus,  staining  by  the  ordinary  methods 
and  by  Gram's  method. 

Although,  as  stated,  Staphylococcus  pyogenes  albus  is 
a  common  cause  of  suppuration,  it  rarely  occurs  alone, 
Passet  so  finding  it  in  but  4  out  of  33  cases  investigated. 
When  pure  cultures  of  the  coccus  are  subcutaneously  in- 
jected into  rabbits  and  guinea-pigs,  abscesses  occasionally 
result.  Injected  into  the  circulation,  the  staphylococci  oc- 
casionally cause  septicemia,  and  after  death  can  be  found  in 
the  capillaries,  especially  in  the  kidneys.  From  this  it  will 
be  seen  that  the  organism  is  feebly  and  variably  pathogenic. 

In  its  morphologic  and  vegetative  characteristics  Sta- 
phylococcus albus  is  almost  identical  with  the  species  next 
to  be  described,  differing  from  it  only  in  the  absence  of  its 
characteristic  golden  pigment. 


STAPHYLOCOCCUS  PYOGENES  AUREUS  (RosENBACHf). 

General  Characteristics. — A  non-motile,  non-flagellate,  non- 
sporogenous,  liquefying,  chromogenic,  pathogenic,  aerobic  and  option- 
ally anaerobic  coccus,  staining  by  the  ordinary  methods  and  by  Gram's 
method. 

Commonly  present  upon  the  skin,  though  in  smaller 
numbers  than  the  organisms  already  described,  is  the  more 
virulent  and  sometimes  dangerous  Staphylococcus  pyogenes 
aureus  (Fig.  102),  or  "golden  Staphylococcus,"  first  observed 
by  Ogston  and  cultivated  by  Rosenbach.  As  the  mor- 
phology and  cultural  characteristics  of  this  organism  are 
identical  with  those  of  the  preceding  species,  it  seems  con- 
venient to  describe  them  together,  pointing  out  such  minor 
differences  as  occur.  In  doing  this,  however,  it  must  not 
be  forgotten  that,  although  Staphylococcus  albus  was  first 
mentioned,  Staphylococcus  aureus  is  the  more  common 
organism  of  suppuration. 

*  "  Wundinf  ektionskrankheiten  des  Menschen,"  Wiesbaden,  1884. 
f  "  Mikroorganismen    bei    Wundinf  ektionskrankheiten     des    Men- 
schen," Wiesbaden,  1884. 


344  Suppuration 


STAPHYLOCOCCI    PYOGENES   AUREUS   ET   ALBUS. 

Distribution. — The  cocci  are  not  widely  distributed  in 
nature,  seeming  not  to  find  a  purely  saprophytic  existence 
satisfactory.  They  occur,  however,  upon  man  and  the 
lower  animals,  and  can  occasionally  be  found  in  the  dusts  of 
houses  and  hospitals — especially  in  the  surgical  wards — if 
proper  precautions  are  not  exercised.  They  are  common 
upon  the  skin,  in  the  nose,  mouth,  eyes,  and  ears  of  man; 
they  are  nearly  always  present  beneath  the  finger-nails, 
and  sometimes  occur  in  the  feces,  especially  of  children. 


Fig.  102. — Staphylococcus  pyogenes  aureus  (Gunther). 

Morphology. — The  cocci  are  small,  measuring  about 
0.7  ^  in  diameter.  When  properly  stained,  the  organisms  are 
found  to  consist  of  hemispheres  separated  from  one  another 
by  a  narrow  interval,  the  approximated  surfaces  being  flat- 
tened. As  observed  in  hastily  stained  preparations,  they 
are  spheric.  There  is  no  definite  grouping  in  either  liquid 
or  solid  cultures.  It  is  only  in  pus  or  in  the  organs  or  tissues 
of  diseased  animals  that  one  can  say  that  a  true  staphylo- 
coccus  grouping  occurs. 

The  organisms  are  not  motile  and  have  no  flagella. 

Staining. — The  organisms  stain  easily  and  brilliantly 
with  aqueous  solutions  of  the  anilin  dyes  and  by  Gram's 
method. 

Isolation. — Staphylococcus  aureus  is  an  easy  organism  to 
isolate,  and  can  be  secured  by  plating  out  a  drop  of  pus  in 


Staphylococci  Pyogenes  Aureus  et  Albus      345 

gelatin  or  in  agar-agar.  Such  preparations,  however,  gen- 
erally do  not  contain  Staphylococcus  aureus  by  itself,  but  in 
association  with  Staphylococcus  albus. 

The  colonies  of  Staphylococcus  aureus  differ  considerably 
in  color,  some  being  much  paler  than  others. 

Cultivation. — The  staphylococci  grow  well  upon  all  the 
standard  culture-media  either  in  the  presence  or  in  the  ab- 
sence of  oxygen  at  temperatures  above  18°  C.,  the  most  rapid 
development  being  at  about  37°  C. 

Colonies. — Upon  the  surface  of  gelatin  plates  the  colo- 
nies appear  as  small  whitish  points  after  from  twenty- 


Fig.  103. — Staphylococcus  pyogenes  aureus.    Colony  two  days  old,  seen 
upon  an  agar-agar  plate.      X  40  (Heim). 


four  to  forty-eight  hours,  rapidly  extending  to  the  sur- 
face and  causing  extensive  liquefaction  of  the  medium. 
The  formation  of  the  orange  pigment  can  be  best  observed 
near  the  center  of  the  colonies.  Under  the  microscope  the 
colonies  appear  as  round  disks  with  circumscribed,  smooth 
edges.  They  are  distinctly  granular  and  dark  brown.  When 
the  colonies  are  grown  upon  agar-agar  plates,  the  formation 
of  the  pigment  is  more  distinct. 

Gelatin  Punctures. — In  gelatin  the  growth  occurs  along 
the  whole  length  of  the  puncture,  causing  an  extensive 
liquefaction  of  the  medium  in  the  form  of  a  long,  narrow, 
blunt-pointed,  inverted  cone,  sometimes  described  as  being 
like  a  stocking,  full  of  clouded  liquid,  at  the  apex  of  which 
a  collection  of  golden  or  orange-yellow  precipitate  is  always 


346 


Suppuration 


present  in  Staphylococcus  aureus.  It  is  this  precipitate  in 
particular  that  gives  the  organism  its  name,  "  golden  sta- 
phylococcus." 

Agar-agar. — The  growth  of  the  golden  Staphylococcus 
upon  agar-agar  is  subject  to  considerable  variation  in  the 
quantity  of  pigment  produced.  Sometimes,  perhaps  rarely, 
it  is  golden;  more  commonly  it  is  yel- 
low, often  cream  color.  Along  the 
whole  line  of  inoculation  a  moist, 
shining,  usually  well-circumscribed 
growth  occurs.  When  the  develop- 
ment occurs  rapidly,  as  in  the  incu- 
bator, it  exceeds  the  rapidity  of  color 
production,  so  that  the  center  of  the 
growth  is  distinctly  colored,  the  edges 
remaining  white. 

Potato. — Upon  potato  the  growth 
is  luxuriant,  Staphylococcus  aureus 
producing  an  orange-yellow  coating 
over  a  large  part  of  the  surface.  The 
potato  cultures  may  give  off  a  sour 
odor. 

Bouillon. — When  grown  in  bouillon 
the  organism  causes  a  diffuse  cloudi- 
ness, with  a  small  quantity  of  slightly 
yellowish  sediment.  The  reaction  of 
the  medium  is  increasingly  alkaline. 
Nitrates  are  reduced  to  nitrites. 

Milk. — In  milk,  coagulation  takes 
place  in  about  eight  days,  and  is  fol- 
lowed by  gradual  digestion  of  the 
casein. 

Thermal  Death  Point. — Staphylococci  are  usually  quite 
susceptible  to  the  effect  of  heat,  though  their  resistance  is 
not  uniform.  Sternberg  found  them  destroyed  by  an 
exposure  to  62°  C.  for  ten  minutes,  and  to  80°  C.  for  one 
and  a  half  minutes,  but  three  cultures  studied  by  von  Lin- 
gelsheim  were  not  killed  by  an  exposure  to  60°  C.  for  an 
hour,  and  one  culture  studied  by  him  endured  an  exposure 
to  80°  C.  for  ten  minutes. 

Toxic  Products. — Leber  seems  to  have  first  conceived  of 
suppuration  as  a  toxic  process  depending  upon  the  soluble 
products  of  parasitic  fungi,  and  in  1888,  through  the  action 


Fig.  104. — Staphylo- 
coccus pyogenes  au- 
reus. Puncture  cul- 
ture three  days  old  in 
gelatin  (Frankel  and 
Pfeiffer). 


Staphylococci  Pyogenes  Aureus  et  Albus      347 

of  alcohol  upon  staphylococci,  prepared  an  acicular  crystal- 
line body  soluble  in  alcohol  and  ether,  but  slightly  soluble 
in  water,  to  which  he  gave  the  name  phlogosin. 

Mannatti  found  that  pus  has  substantially  the  same  toxic 
properties  as  sterilized  cultures  of  the  staphylococcus ;  that 
repeated  injections  of  sterilized  pus  induce  chronic  in- 
toxication and  marasmus;  that  injection  of  sterilized  pus 
under  the  skin  causes  a  grave  form  of  poisoning ;  and  that 
the  symptoms  and  pathologic  lesions  caused  by  these 
injections  correspond  with  those  observed  in  men  suffering 
from  chronic  suppuration. 

Van  de  Velde*  found  that  the  staphylococcus  has  some 
metabolic  products  destructive  to  the  leukocytes,  which 
he  has  called  leukocidin.  This  poison  causes  the  cells  to 
cease  ameboid  movement,  become  spheric,  and  gradually 
to  lose  their  granules,  until  they  finally  appear  like  empty 
sacs  containing  shadow  nuclei,  which  eventually  disappear. 
The  leukolysis  occurs  in  about  two  minutes.  These  ob- 
servations have  been  abundantly  confirmed.  Kraussf  first 
observed  that  certain  products  of  the  staphylococcus  were 
hemolytic  and  destroyed  red  blood-corpuscles.  This  hemo- 
lysin  has  been  carefully  studied  by  Neisser  and  Wechsberg,  J 
by  whom  it  was  called  staphylolysin. 

Durme§  found  staphylolysin  produced  most  abundantly 
by  virulent  staphylococci. 

Ribbertll  found  that  both  sterilized  and  unsterilized  cul- 
tures when  intravenously  injected  into  animals  produced 
definite  changes  in  the  heart,  kidneys,  lungs,  spleen,  and 
bone-marrow,  and  attributed  the  action  to  the  toxin. 

Morse**  found  that  the  toxic  products  of  Staphylococcus 
aureus  were  capable  of  occasioning  interstitial  nephritis. 

The  staphylococci  form  very  little  extracellular  toxin,  as 
filtered  cultures  provoke  little  local  or  general  reaction  in 
animals,  even  when  the  staphylococcus  is  highly  virulent. 

Pathogenesis. — The  virulent,  golden  staphylococcus  is 
a  dangerous  and  often  deadly  organism.  Its  virulence  is, 

"La  Cellule,"  xi,  1896,  p.  349. 
t  "Wiener,  klin.  Wochenschrift,"  1900. 
|  "Zeitschrift  fur  Hygiene,"  1911,  xxxvi,  p.  330. 
§  "Hyg.  Rundschau,"  1903,  Heft  2,  p.  66. 

||  "Die   pathologische   Anatomie  und   die   Heilung   der  durch   den 
Staphylococcus  pyogenes  aureus  hervorgerufenen  Erkrankungen." 
**  "Journal  of  Experimental  Medicine,"  vol.  I,  1896,  p.  613. 


348  Suppuration 

however,  very  variable  both  for  the  lower  animals  and  for 
man.  The  classical  test  for  virulence  is  to  inject  TV  c.c.  of 
a  twenty-four-hour  old  bouillon  culture  into  the  ear  vein  of  a 
middle-sized  rabbit.  If  of  the  ordinary  virulence,  the 
organism  should  kill  the  rabbit  in  from  four  to  eight  days. 
During  this  time  the  animal  suffers  from  fever  and  wasting, 
and  when  examined  postmortem  almost  invariably  shows 
small  abscesses  in  the  kidneys  and  heart.  In  cases  in  which 
the  rabbits  are  highly  susceptible  or  the  cocci  virulent, 
purulent  arthritis  may  be  found.  Highly  virulent  cultures 
kill  the  animal  in  from  one  to  two  days,  commonly  by 
occasioning  endocarditis. 

When  the  cocci  enter  human  beings  subcutaneously,  ab- 
scesses commonly  result,  and  occasionally  lead  to  a  fatal 
generalization  of  the  organisms.  In  such  cases  the  organisms 
may  be  cultivated  from  the  streaming  blood,  though  the 
greater  number  collect  in,  and  frequently  obstruct,  the  capil- 
laries. In  the  lungs  and  spleen,  and  still  more  frequently  in 
the  kidneys,  infarcts  are  formed  by  the  bacterial  emboli. 
The  Malpighian  tufts  of  the  kidneys  are  sometimes  full  of 
cocci,  and  become  the  centers  of  small  abscesses. 

The  coccus  is  almost  equally  pathogenic  for  man  and  the 
lower  animals,  though  the  fatal  outcome  of  human  infection 
is  more  rare,  possibly  because  of  the  conditions  of  infection. 
It  enters  the  human  system  through  scratches,  punctures, 
or  abrasions,  and  when  virulent  usually  occasions  an  ab- 
scess. Garre*  applied  the  organism  in  pure  culture  to  the 
uninjured  skin  of  his  arm,  and  in  four  days  developed  a  large 
carbuncle,  with  a  surrounding  zone  of  furuncles.  Bockhartf 
suspended  a  small  portion  of  an  agar-agar  culture  in  salt 
solution,  and  scratched  it  gently  into  the  deeper  layer  of  the 
skin  with  his  finger-nail;  a  furuncle  developed.  Bumm 
injected  the  coccus  suspended  in  salt  solution  beneath  his 
skin  and  that  of  several  other  persons,  and  produced  an 
abscess  in  every  case. 

Staphylococcus  aureus  is  not  only  found  in  the  great 
majority  of  furuncles,  carbuncles,  abscesses,  and  other  in- 
flammatory diseases  of  the  surface  of  the  body,  but  also  plays 
an  important  role  in  a  number  of  deeply  seated  diseases. 
Becker  and  others  obtained  it  from  the  pus  of  osteomyelitis, 
demonstrating  that  if,  after  fracturing  or  crushing  a  bone, 

*  "Fortschritte  der  Med.,"  1885,  No.  6. 

f  "Monatschrift  fur  prakt.  Dermatologie,"  1887,  iv,  No.  10. 


Staphylococci  Pyogenes  Aureus  et  Albus      349 

the  staphylococcus  be  injected  into  the  circulation,  osteo- 
myelitis may  occur.  Numerous  observers  have  demon- 
strated its  presence  in  ulcerative  endocarditis.  Rodet  has 
been  able  to  produce  osteomyelitis  without  previous  injury 
to  the  bones;  Rosenbach  was  able  to  produce  ulcerative 
endocarditis  by  injecting  some  of  the  staphylococci  into  the 
circulation  in  animals  whose  cardiac  valves  had  been  injured 
by  a  sound  passed  into  the  carotid  artery;  and  Ribbert  has 
shown  that  the  injection  of  cultures  of  the  organism  may 
cause  valvular  lesions  without  preceding  injury. 

Virulence. — Experiments  have  shown  that  both  Staphy- 
lococci aureus  and  albus  exist  in  attenuated  and  virulent 
forms,  and  there  is  every  reason  to  believe  that  in  the  major- 
ity of  instances  they  inhabit  the  surface  of  the  body  in  a 
feebly  virulent  condition. 

Agglutination.— Kolle  and  Otto*  have  found  that  im- 
mune antistaphylococcic  serums  agglutinate  the  staphylo- 
cocci. The  reaction  is  not  specific  and  is  peculiar.  All 
pathogenic  staphylococci  are  agglutinated;  non-pathogenic 
cocci  are  not  agglutinated.  The  reaction  cannot,  therefore, 
be  used  for  specific  differentiation. 

Specific  Therapy. — The  treatment  of  staphylococcus  in- 
fections with  immune  serum  has  not  met  with  encouraging 
success.  Viqueratf  has  experimented  in  this  direction  and 
found  that  goats  are  best  adapted  to  the  manufacture  of 
the  serum ;  but  the  literature  of  medicine  contains  very  little 
mention  of  beneficial  results  following  the  employment  of 
antistaphylococcus  serums. 

Denys  and  van  de  Veldet  and  Neisser  and  Wechsberg§ 
also  produced  antileukocytic  serum. 

Bacterio= vaccination. — Although  specific  serums  have 
failed,  a  promising  form  of  specific  treatment  for  subacute 
and  chronic  staphylococcic  infections  has  been  introduced  by 
A.  B.  Wright,  ||  who  first  isolates  from  the  lesion  the  partic- 
ular strain  of  staphylococci  by  which  it  is  caused,  cultivates 
this  artificially,  suspends  the  organisms  in  an  indifferent 
fluid,  of  which  a  given  quantity  contains  a  known  (counted) 

*  "Zeitschrift  fur  Hygiene,"  etc.,  1902,  xu. 
t  Ibid.,  xvm,  1894,  p.  483. 
}  "La  Cellule,"  1895,  xi. 
§  "  Zeitschrift  fur  Hygiene,"  1901,  xxxn. 

||  "Lancet,"  March  29,  1902,  p.  874;  "Brit.  Med.  Jour.,"  May  9, 
1903,  p.  1069. 


35°  Suppuration 

number,  kills  the  organisms  by  heating  them  for  an  hour  at 
60°  C.,  and  then  uses  them  by  subcutaneous  injection  for 
producing  increased  resistance  on  the  part  of  the  patient. 

The  treatment  is  controlled  by  studying  the  "  opsonic 
index  "  (q.v.),  the  objects  being  the  avoidance  of  the  "  nega- 
tive phase  "  or  condition  of  diminished  resistance,  and  the 
progressive  establishemnt  of  the  positive  phase  or  stage  of 
increased  resistance.  As  the  resistance  increases  the  patient 
rapidly  improves,  and  many  cases  of  obstinate  acne,  furun- 
culosis,  and  other  pyogenic  infections  have  quickly  recovered 
under  this  treatment. 


STAPHYUDCOCCUS  CITREUS  (PASSET). 

An  organism  identical  in  many  respects  with  the  pre- 
ceding, except  that  its  growth  on  agar-agar  and  potato  is 
of  a  brilliant  lemon-yellow  color  and  its  pathogenicity  for 
animals  doubtful,  is  Staphylococcus  citreus  of  Passe t.*  As 
it  is  not  common  and  is  doubtfully  pathogenic,  it  is  of  much 
less  importance  than  the  previously  described  organisms. 


STREPTOCOCCUS  PYOGENES  (ROSENBACH). 

General  Characteristics.— The  streptococcus  is  a  non-motile,  non- 
flagellate,  non-sporogenous,  non-liquefying,  non-chromogenic,  aerobic 
and  optionally  anaerobic,  spheric  organism,  infectious  for  man  and  the 
lower  animals.  Its  division  in  one  direction  of  space  leads  to  its  asso- 
ciation in  the  form  of  chains  or  "strings  of  beads."  It  stains  by  ordi- 
nary methods  and  by  Gram's  method. 

Streptococci  were  probably  first  seen  by  Kochf  in  1878. 
In  1 88 1  OgstonJ  called  attention  to  the  fact  that  two  distinct 
kinds  of  cocci  were  to  be  found  in  pus,  mentioning  both 
staphylococci  and  streptococci.  The  beginning  of  real 
knowledge  of  the  streptococci,  however,  dates  from  the  time 
of  their  isolation  and  cultivation  by  Fehleisen§  and  of 
Rosenbach,  ||  who  cultivated  them  from  1 8  of  33  suppurative 

"  Untersuchungen  iiber  die  Aetiologie  der  eitrigen  Phlegmone  des 
Menschen,"  Berlin,  1885,  P-  9- 

t  "  Untersuchungen  iiber  die  Aetiologie  der  Wundinfektionskrank- 
heiten,"  Leipzig,  Vogel,  1878.  , 

t  "British  Med.  Jour.,"  March,  1881,  p.  369. 

§  "Aetiologie  des  Erysipels,"  Berlin,  Fischer,  1883. 

||  "Mikroorganismen  bei  Wundinfektionskrankheiten  des  Menschen," 
1884,  p.  22. 


Streptococcus  Pyogenes  351 

lesions,  fifteen  times  alone  and  five  times  in  association 
with  Staphylococcus  aureus. 

Morphology. — The  organisms  are  spheric,  of  variable  size 
(0.4-1  [i  in  diameter),  and  are  constantly  associated  in  pairs 
or  in  chains  of  from  four  to  twenty  or  more  individuals. 
Special  varieties,  known  as  Streptococcus  longus  (chains  of 
more  than  one  hundred  members)  and  Streptococcus  brevis 
(chains  of  from  four  to  ten),  have  been  described  by  v. 
Lingelsheim,*  but  do  not  hold  as  separate  species. 

It  is  not  motile  and  does  not  form  endospores,  though 
sometimes  large  individuals — much  larger  than  the  others  in 


Fig.  105. — Streptococcus  pyogenes,  from  the  pus  taken  from  an  ab- 
scess.     X   looo  (Frankel  and  Pfeiffer). 

the  chain — may  be  observed.  Some  believe  these  to  be 
arthrospores. 

Staining. — The  organisms  stain  well  with  ordinary 
aqueous  solutions  of  anilin  dyes  and  by  Gram's  method. 

Isolation. — The  streptococcus  can  be  isolated  from  pus 
containing  it  by  plating  or  by  the  inoculation  of  a  mouse  or 
rabbit,  from  whose  blood  it  may  easily  be  secured  after 
death. 

Cultivation. — The  organism  grows  at  both  the  room  tem- 
perature and  that  of  incubation,  its  best  and  most  rapid 
development  being  at  about  37°  C. 

*  "Zeitschrift  fur  Hygiene,"  Bd.  x,  1891,  p.  331;  xn,  1892,  p.  308. 


352  Suppuration 

Colonies. — Upon  gelatin  plates  very  small,  colorless,  trans- 
lucent colonies  appear  in  from  twenty-four  to  forty-eight 
hours.  When  superficial,  they  spread  out  to  form  flat  disks 
about  0.5  mm.  in  diameter.  The  microscope  shows  them  to 
be  irregular  and  granular,  to  have  a  slightly  yellowish  color 
by  transmitted  light,  and  to  have  numerous  irregularities 
around  the  edges,  due  to  projecting  chains  of  the  cocci.  No 
liquefaction  of  the  gelatin  occurs. 

Gelatin  Punctures. — In  gelatin  puncture  cultures  no 
liquefaction  is  observed.  The  minute  spheric  colonies  grow 


Fig.  106. — Streptococcus  colonies  on  serum  agar  (From  Hiss  and  Zins- 
ser,  '•  Text-Book  of  Bacteriology,"  D.  Appleton  &  Co.,  Publishers). 


along  the  whole  length  of  the  puncture  and  form  a  slightly 
opaque  granular  line. 

Agar-agar. — Upon  agar-agar  an  exceedingly  delicate 
transparent  growth  develops  slowly  along  the  line  of  inocu- 
lation. It  consists  of  small,  colorless,  or  slightly  grayish 
transparent  colonies  which  do  not  readily  coalesce. 

Blood-serum. — The  growth  upon  blood-serum  resembles 
that  upon  agar-agar.  The  colonies  are  small,  white,  dis- 
crete, and  do  not  affect  the  medium. 

Potato. — The  streptococcus  does  not  seem  to  grow  well 
upon  potato,  the  colonies  being  invisible. 

Bouillon. — In  bouillon  the  cocci  develop  slowly,  seeming 
to  prefer  a  neutral  or  feebly  alkaline  reaction.  The  medium 
remains  clear,  while  numerous  small  flocculi  are  suspended 
in  it,  sometimes  adhering  to  the  sides  of  the  tube,  sometimes 
forming  a  sediment.  When  the  flocculi  formation  is  distinct, 
the  name  Streptococcus  conglomerate  (Kurth)  is  sometimes 
given  to  the  organism ;  when  the  medium  is  diffusely  clouded, 
it  is  called  Streptococcus  dijjusus. 


Streptococcus  Pyo genes  353 

In  mixtures  of  bouillon  and  blood-serum  or  ascitic  fluid 
the  streptococcus  grows  more  luxuriantly,  especially  at  in- 
cubation temperatures,  distinctly  clouding  the  liquid.  As 
the  lactic  acid  which  is  rapidly  formed  inhibits  the  growth 
of  the  cocci,  Hiss  recommends*  that  instead  of  eliminating 
the  sugars  in  the  broth,  upon  which  the  streptococci  are 
nourished,  i  per  cent,  of  sterile  powdered  CaCO3  be  added 
to  the  culture-media.  This  neutralizes  the  acid  as  rapidly  as 
it  is  formed.  It  also  maintains  the  life  of  the  culture  for  a 
long  time. 

Milk. — The  organism  seems  to  grow  well  in  milk,  which  is 
coagulated  and  digested. 

Reaction. — The  streptococcus  is  sensitive  to  acids,  and 
can  only  grow  well  in  media  with  a  slightly  acid  reaction. 
All  streptococci  produce  acids  and  eventually  acidulate  the 
media,  thus  checking  their  further  development. 

Vital  Resistance. — The  optimum  temperature  appears  to 
be  in  the  neighborhood  of  37°  C.  It  grows  well  between 
25°  and  40°  C.,  above  40.5°  C.  the  growth  is  slowed.  The 
thermal  death  point  is  low.  Sternberg  found  that  the 
streptococci  succumb  at  temperatures  of  52°  to  54°  C.  if 
maintained  for  ten  minutes.  Their  vitality  in  culture  is 
slight,  and  unless  frequently  transplanted  they  die.  Bouil- 
lon cultures  usually  die  in  from  five  to  ten  days.  On  solid 
media  they  seem  to  retain  their  vegetative  and  pathogenic 
powers  much  longer,  especially  if  kept  cool  and  cultivated 
beneath  the  surface  of  the  medium  in  a  deep  puncture.  They 
resist  drying  well. 

Differential  Features. — It  is  not  always  easy  to  dif- 
ferentiate Streptococcus  pyogenes  from  other  less  impor- 
tant forms  of  streptococci  and  from  the  pneumococcus. 
One  of  the  best  methods  is  by  the  employment  of  blood-agar 
plates,  suggested  by  Schottmuller.t  Such  plates  are  easily 
prepared  by  melting  ordinary  culture  agar-agar,  cooling  to 
about  45°  C.,  and  then  adding  about  0.5  c.c.  of  defibrinated 
human  or  rabbit's  blood  to  the  tube.  The  blood  is  first 
thoroughly  mixed  with  the  agar,  then  the  tube  inoculated, 
and  poured  into  a  Petri  dish.  As  the  Streptococcus  pyogenes 
grows,  it  produces  a  hemolytic  substance  that  destroys  the 
blood-corpuscles  in  the  vicinity  of  the  colony,  thus  sur- 

*" Text-book  of  Bacteriology,"  p.  338. 

f  "Munch,  med.  Wochenschrift, "  1903,  L,  p.  909. 

23 


354  Suppuration 

rounding  each  by  a  clear,  pale  halo  that  contrasts  with  the 
red  agar.  The  colonies  themselves  appear  gray. 

The  test  is  not  specific,  and  Ruediger*  points  out  that  the 
diphtheria  and  pseudodiphtheria  bacilli  also  produce 
hemolyzing  substance,  so  that  the  test  cannot  be  used  for 
the  immediate  separation  of  streptococci  from  other  bacteria 
in  cultures  from  the  throat.  Colonies  of  the  pneumococcus 
usually  appear  green  and  without  hemolysis,  but  Ruediger 
finds  that  they  sometimes  also  cause  solution  of  the  hemo- 
globin. The  streptococci  whose  colonies  are  green  and 
without  hemolysis  are  called  Streptococcus  viridans  by  Schott- 
muller.  They  are  practically  without  pathogenic  power 
for  rabbits. 

Pathogenesis. — The  streptococcus  has  been  found  in 
erysipelas,  ulcerative  endocarditis,  periostitis,  otitis,  men- 
ingitis, empyema,  pneumonia,  lymphangitis,  phlegmons, 
sepsis,  puerperal  endometritis,  and  many  other  forms  of 
inflammation  and  septic  infection.  In  man  it  is  usually 
associated  with  active  forms  of  suppuration  and  sepsis. 

The  relation  of  the  streptococcus  to  diphtheria  is  of 
interest,  for,  though  in  all  probability  the  great  majority  of 
cases  of  pseudomembranous  angina  are  caused  by  the 
Klebs-Loffler  bacillus,  yet  a  number  are  met  with  in  which, 
as  in  Prudden's  24  cases,  no  diphtheria  bacilli  can  be  found, 
but  which  seem  to  be  caused  by  the  streptococcus. 

There  is  no  clinical  difference  between  the  throat  lesions 
produced  by  the  two  organisms,  and  the  only  positive 
method  of  differentiating  the  one  from  the  other  is  by  means 
of  a  careful  bacteriologic  examination.  Such  an  examina- 
tion should  always  be  made,  as  it  has  much  weight  in  con- 
nection with  the  treatment;  in  streptococcus  angina  no 
benefit  can  be  expected  from  the  administration  of  diphtheria 
antitoxic  serum. 

Hirshf  has  shown  that  streptococci  are  by  no  means  rare 
in  the  intestines  of  infants,  where  they  may  occasion  enter- 
itis. In  such  cases  the  organisms  are  found  in  large  num- 
bers in  the  stomach  and  in  the  stools,  and  late  in  the  course 
of  the  disease  in  the  blood  and  urine  of  the  child.  They 
also  occur  in  all  of  the  internal  organs  of  the  cadaver. 

The  intestinal  streptococci  are  often  Gram-negative,  when 
they  are  usually  non-virulent. 

*  "Jour.  Amer.  Med.  Assoc.,"  1906,  XLVII,  p.  1171. 

t  "Centralbl.  f.  Bakt.  u.  Parasit.,"  Bd.  xxn,  Nos.  14  and  15,  p.  369. 


Streptococcus  Pyogenes  355 

Libman*  has  reported  2  carefully  studied  cases  of  strep- 
tococcic  enteritis. 

Flexner,  f  in  a  large  series  of  autopsies,  found  the  bodies 
invaded  by  numerous  micro-organisms,  causing  what  he  has 
called  "  terminal  infections,"  and  hastening  the  fatal  issue. 
Of  793  autopsies  at  the  Johns  Hopkins  Hospital,  255  upon 
cases  dying  of  chronic  heart  or  kidney  diseases,  or  both,  were 
sufficiently  well  studied,  bacteriologically,  to  meet  the  re- 
quirements of  a  statistical  inquiry.  Tuberculous  infections 
were  not  included.  Of  the  255  cases,  213  gave  positive 
bacteriologic  results.  '  The  micro-organisms  causing  the  in- 
fections, 38  in  all,  were  Streptococcus  pyogenes,  16  cases; 
Staphylococcus  pyogenes  aureus,  4  cases;  Micrococcus  lan- 
ceolatus,  6  cases;  gas  bacillus  (Bacillus  aerogenes  capsu- 
latus),  three  times  alone  and  twice  combined  with  B.  coli 
communis;  the  gonococcus,  anthrax  bacillus,  B.  proteus,  the 
last  combined  with  B.  coli;  B.  coli  alone;  a  peculiar  capsu- 
lated  bacillus,  and  an  unidentified  coccus." 

It  is  interesting  to  observe  in  how  many  cases  the  strepto- 
coccus was  present.  All  the  streptococci  found  may  not 
have  been  Streptococcus  pyogenes,  but  for  convenience 
in  his  statistics  they  were  regarded  as  such. 

The  presence  of  streptococci  in  the  blood  in  scarlatina  has 
been  observed  in  30  cases  by  Crooke,  by  Frankel  and  Tren- 
denburg,  Raskin,  Leubarth,  Kurth,  and  Babes.  In  1 1  cases 
of  scarlatina  studied  by  Wright  {  a  general  streptococcus 
infection  occurred  in  4,  a  pneumococcus  infection  in  i,  and  a 
mixed  infection  of  pyogenic  cocci  in  i. 

Lemoine§  found  streptococci  in  the  blood  during  life  in  2 
out  of  33  cases  of  scarlet  fever  studied.  Pearcell  studied  17 
cases  of  scarlatina  and  found  streptococci  in  the  heart's 
blood  and  liver  in  4,  in  the  spleen  in  2,  in  the  kidney  in  5 
cases.  In  2  of  the  cases  Staphylococcus  pyogenes  aureus 
was  associated  with  the  streptococcus. 

The  streptococcus  is  the  most  common  organism  found  in 
the  suppurative  sequelae  of  scarlatina,  frequently  occurring 
alone;  sometimes  with  the  staphylococci ;  sometimes  with 
the  pneumococci.  Councilman  found  secondary  infection 

*  "Centralbl.  f.  Bakt.  u.  Parasit.,"  Bd.  xxii,  Nos.  14  and  15,  p.  376. 

f  "Journal  of  Experimental  Medicine,"  vol.  I,  No.  3,  1896. 

J  "Boston  Med.  and  Surg.  Jour.,"  March  21,  1895. 

§  "Bull,  et  Mem.  Soc.  d'Hop.  de  Paris,"  1896,  3  s.,  xm. 

||  "Jour.  Boston  Soc.  of  Med.  Sci.,"  March,  1898. 


356  Suppuration 

by  the  streptococcus  more  widespread  in  variola  than  in 
any  other  disease. 

Virulence. — In  the  great  majority  of  cases,  streptococci 
isolated  from  human  beings  are  pathogenic  for  rabbits  and 
mice.  Rats  become  ill  when  injected  with  large  doses,  but 
usually  recover.  Guinea-pigs,  cats,  and  dogs  are  but 
slightly  susceptible.  Large  animals,  like  sheep,  goats,  cattle, 
and  horses,  react  very  slightly  to  large  doses,  but  sometimes 
suffer  from  abscesses  at  the  seat  of  injection.  Mice  die  in 
from  one  to  four  days  from  general  infection.  If  the  organ- 
isms are  less  virulent,  they  die  in  from  four  to  six  days  with 
edema  and  abscess  formation  at  the  site  of  inoculation,  and 
subsequent  invasion  of  the  body.  The  streptococcus  seems 
to  be  most  pathogenic  for  that  species  of  animal  from  which 
it  has  been  isolated. 

If  the  ear  of  a  rabbit  be  carefully  inoculated  with  a  small 
quantity  of  a  pure  culture,  local  erysipelas  usually  results, 
the  disturbance  passing  away  in  a  few  days  and  the  animal 
recovering.  If,  however,  the  streptococcus  be  highly  viru- 
lent, the  rabbit  dies  of  general  septicemia  in  from  twenty- 
four  hours  to  six  days.  The  cocci  may  then  be  found  in 
large  numbers  in  the  heart's  blood  and  in  the  organs.  In 
less  virulent  cases  minute  disseminated  pyemic  abscesses  are 
sometimes  found. 

According  to  Marmorek,*  the  virulence  of  the  strepto- 
coccus can  be  increased  to  a  remarkable  degree  by  rapid 
passage  through  rabbits,  and  maintained  by  the  use  of  a  cul- 
ture-medium consisting  of  3  parts  of  human  blood-serum 
and  i  of  bouillon.  The  blood  of  the  ass  or  ascitic  or  pleu- 
ritic exudates  may  be  used  instead  of  the  human  blood-serum 
if  the  latter  be  unobtainable.  By  these  means  Marmorek 
succeeded  in  intensifying  the  virulence  of  a  culture  to  such 
a  degree  that  one  hundred-thousand-millionth  (un  cent  mil- 
liardieme)  of  a  cubic  centimeter  injected  into  the  ear  vein 
was  fatal  to  a  rabbit. 

Petruschkyf  found  the  virulence  of  the  culture  to  be  well 
retained  when  the  organisms  were  planted  in  gelatin,  trans- 
planted every  five  days,  and  when  grown,  kept  on  ice. 

Hoist  J  observed  a  virulent  Streptococcus  brevis  that  re- 

*  "Ann.  de  1'Inst.  Pasteur,"  t.  ix,  No.  7,  July  25,  1895,  p.  593. 
t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Bd.  xvm,  No.  16,  May  4, 
1895,  P-  55i. 

%  Ibid.,  Bd.  xix,  No.  n,  March  21,  1896 


Streptococcus  Pyogenes  357 

mained  unchanged  upon  artificial  culture -media  for  eight 
years  without  any  particular  precautions  having  been  taken 
to  maintain  the  virulence. 

Dried  streptococci  are  said  by  Frosch  and  Kolle  *  to  retain 
their  virulence  longer  than  those  growing  on  culture-media. 

Marmorek  f  and  Lubenau  J  found  that  cultures  of  the  strep- 
tococcus when  grown  in  bouillon  containing  glucose,  produced 
a  hemolytic  substance — streptokolysin — not  seemingly  pres- 
ent in  cultures  grown  in  ordinary  bouillon.  Besredka§ 
found  that  streptokolysin  was  produced  only  by  highly  viru- 
lent cultures  of  the  streptococcus  and  not  by  saprophytic 
organisms  that  have  been  for  some  time  under  cultivation  in 
the  laboratory. 

Levin  |1  investigated  the  subject  thoroughly  and  found 
that  different  strains  of  streptococci  produced  strepto- 
kolysin in  varying  quantities,  that  its  production  is  entirely 
independent  of  virulence,  that  it  is  destroyed  by  heat 
(37°  C.  in  some  days;  55°  C.  in  one-half  hour);  that  acidity 
of  the  nutrient  media  hinders  its  formation,  and  that  it  is 
intimately  associated  with  the  bodies  of  the  streptococci 
by  which  it  is  produced,  so  that  in  the  sediment  obtained  by 
filtration  or  by  centrifugation  there  is  nearly  one  thousand 
times  as  much  as  in  the  filtered  fluid  culture.  The  strepto- 
kolysin is  not  destroyed  by  the  death  of  the  bacteria.  An- 
tistreptokolysin  is  present  in  antistreptococcus  serum. 

Toxic  Products. — The  toxic  products  of  the  strepto- 
coccus are  not  well  known.  Cultures  from  different  sources 
vary  greatly  in  the  effects  produced  by  hypodermic  or  intra- 
venous injection  after  filtration  through  porcelain.  Killed 
cultures  produce  a  much  more  marked  effect  than  filtered 
ones,  so  that  the  important  product  must  be  an  endotoxin. 

Simon**  found  that  the  toxic  quality  of  the  bodies  of 
streptococci  of  different  stocks  had  nothing  to  do  with  their 
virulence.  Simon  ft  also  found  that  the  toxic  products  of 
the  streptococcus  were  diverse  and  peculiar.  The  bodies 
of  the  cocci  contained  an  intracellular  toxin  the  activity  of 

*  Flugge's  "Die  Mikroorganismen." 

t  "Annales  de  1'Inst.  Pasteur,"  1895,  593. 

t  "Centralbl.  f.  Bakt.,"  etc.,  1901,  Bd.  xxx,  Nos.  9  and  10. 

§  "Ann.  de  1'Inst.  Pasteur,"  1901,  p.  880. 

||  "Nord.  Med.  Ark,"  1903,  n,  No.  15,  p.  20. 
**  "Centralbl.  f.  Bakt.,"  xxxv,  No.  3,  p.  308,  Dec.  18,  1903. 
tt  Ibid.,  xxxv,  No.  4,  p.  350,  Jan.  16,  1904. 


358  Suppuration 

which  was  independent  of  their  virulence.  This  poison  is 
liberated  only  when  the  bactericidal  activities  of  the  body 
act  upon  the  cocci.  The  cocci  also  excrete  a  toxic  substance 
whose  activity  is  greater  than  that  of  the  intracellular  toxin, 
but  whose  production  is  subject  to  great  variation  and  is 
entirely  independent  of  the  intracellular  toxin.  The  toxins 
and  hemolysins  are  entirely  different  bodies. 

In  general,  the  effects  of  streptococcus  intoxication  are 
vague.  The  animals  appear  weak  and  ill,  and  have  a 
slight  fever;  but  unless  the  virulence  of  the  culture  be 
exceptional  or  the  dose  very  large,  they  usually  recover  in 
a  short  time. 

Coley 's  Mixture. — The  clinical  observation  that  occa- 
sional accidental  erysipelatous  infection  of  malignant  tumors 
is  followed  by  sloughing  and  the  subsequent  disappearance 
of  the  tumor,  suggested  the  experimental  inoculation  of 
such  tumors  with  Streptococcus  erysipelatis  as  a  therapeutic 
measure.  The  danger  of  the  remedy,  however,  caused  many 
to  refrain  from  its  use,  for  when  one  inoculates  the  living 
erysipelas  virus  into  the  tissues  it  is  impossible  to  estimate 
the  exact  amount  of  disturbance  that  will  follow. 

To  overcome  this  difficulty  Coley*  has  recommended 
that  the  toxin  instead  of  the  living  coccus  be  used  for  in- 
jection. A  virulent  culture  of  the  streptococcus  is  obtained, 
by  preference  from  a  fatal  case  of  erysipelas,  inoculated  into 
small  flasks  of  slightly  acid  bouillon,  and  allowed  to  grow 
for  three  weeks.  The  flask  is  then  reinoculated  with  Bacil- 
lus prodigiosus,  allowed  to  grow  for  ten  or  twelve  days  at  the 
room  temperature,  well  shaken  up,  poured  into  bottles  of 
about  f5ss  capacity,  and  rendered  perfectly  sterile  by  an 
exposure  to  a  temperature  of  50°  to  60°  C.  for  an  hour.  It 
is  claimed  that  the  combined  products  of  the  streptococcus 
of  erysipelas  and  Bacillus  prodigiosus  are  much  more  active 
than  a  simple  streptococcus  culture.  The  best  effects  follow 
the  treatment  of  cases  of  inoperable  spindle-cell  sarcoma, 
where  the  toxin  sometimes  causes  a  rapid  necrosis  of  the 
tumor  tissue,  which  can  be  scraped  out  with  an  appropriate 
instrument.  Numerous  cases  are  on  record  in  which  this 
treatment  has  been  most  efficacious;  but,  although  Coley 
still  recommends  it  and  Czerny  upholds  it,  the  majority  of 
surgeons  have  failed  to  secure  the  desired  results. 

*  "Amer.  Jour.  Med.  Sci.,"  July,  1894. 


Streptococcus  Mucosus  359 

Antistreptococcus  Serum. — Since  1895  considerable  at- 
tention has  been  bestowed  upon  the  antistreptococcus  serum 
of  Marmorek*  and  Gromakowsky,  f  which  is  said  to  act 
specifically  upon  streptococcus  infections,  both  general  and 
local.  Numerous  cases  of  suppuration,  septic  infection, 
puerperal  fever,  and  scarlatina  are  upon  record  in  which 
the  serum  seems  to  have  exerted  a  beneficial  action,  and  it 
may  be  that  antiphlogistic  serums  will  occupy  an  important 
place  in  the  medicine  of  the  future. 

The  serum  is  prepared  by  the  injection  of  cultures  of 
living  virulent  streptococci,  into  horses,  until  a  high  degree 
of  immunity  is  attained.  The  serum  is  probably  both 
antitoxic  and  bactericidal  in  action. 

The  success  following  the  serums  of  some  experimenters 
upon  certain  cases,  and  their  occasional  or  constant  failure 
in  other  cases,  have  suggested  that  there  is  considerable 
difference  between  different  "  strains  "  or  families  of  strep- 
tococci. To  obviate  this  inequality  Van  de  VeldeJ  has  made 
a  polyvalent  antistreptococcus  serum  by  using  a  number 
of  different  cultures  secured  from  the  most  diverse  clinical 
cases  of  streptococcus  infection.  Another  serum,  of  Tavel§ 
and  Moser,  ||  is  made  by  using  cultures  from  different  cases 
of  scarlatina.  The  use  of  these  serums,  however,  has  not 
given  the  satisfaction  expected,  and  at  the  present  moment 
the  whole  subject  of  antistreptococcus  serums  is  debatable 
both  from  the  standpoint  of  its  theoretic  scientific  basis 
and  its  therapeutic  application. 

STREPTOCOCCUS  Mucosus  (HOWARD  AND  PERKINS). 

This  organism,  described  by  Howard  and  Perkins,**  was 
isolated  from  a  case  of  tubo-ovarian  abscess  with  generalized 
infection,  and  again  later  by  Schottm  tiller  ft  from  a  case 
of  parametritis,  peritonitis,  meningitis,  and  phlebitis. 

It  occurs  as  a  rounded  coccus  in  pairs  and  in  short  chains, 
though  sometimes  long  chains  of  a  hundred  were  observed. 

*  "Ann.  de  1'Inst.  Pasteur,"  t.  ix,  No.  7,  July  25,  1895,  p.  593. 

t  Ibid. 

J  "Archiv.  de.  med.  Exper.,"  1897. 

§  "Deutsche  med.  Wochenschrift,"  1903,  No.  50. 

||  "Berliner  klin.  Wochenschrift,"  1902,  13. 
**  "Journal  of  Medical  Research,"  1901,  N.  S.  I,  163. 
ft  "Munch,  med.  Wochenschrift,"   1903,  xxi. 


360  Suppuration 

The  pairs  resemble  gonococci.  They  measure  i .  25  to  i .  75^  in 
length  and  0.5  to  0.75  ^  in  breadth.  Each  is  surrounded  by 
a  halo  that  varies  in  width  from  1.5  to  3.0  (/,  which  shows 
best  in  cultures  grown  on  human  blood-serum.  The  usual 
capsule  stains  fail  to  color  this  halo  when  the  organisms  are 
from  artificial  cultures,  though  they  show  it  well  when  they 
are  in  pus.  The  organisms  stain  with  ordinary  dyes  and  by 
Gram's  method. 

The  cultures  resemble  those  of   Streptococcus  pyogenes, 
but  are  rather  more  luxuriant,  the  colonies  having  a  bluish 


*   • 


/  - 


Fig.  107. — Streptococcus  mucosus,  from  peritoneal  exudate.      X  1200 
(Howard  and  Perkins,  in  "  Journal  of  Medical  Research  "). 

cast.  The  organism  ferments  inulin,  which  makes  Hiss  think 
it  related  to  the  pneumococcus. 

The  organism  taken  at  autopsy  and  inoculated  into  the 
peritoneum  of  a  guinea-pig  caused  the  animal  to  die,  coma- 
tose, in  thirty-six  hours  with  peritonitis.  There  were  15  to 
20  c.c.  of  peculiar  viscid  fluid  in  the  peritoneal  cavity.  It 
had  a  grayish  purulent  character  and  contained  numerous 
flakes  of  fibrin.  There  was  no  generalized  infection.  Mice 
and  rabbits  were  susceptible  and  died  of  generalized  in- 
fection. 

The  organism  is  not  infrequently  found  as  an  apparently 
harmless  tenant  of  the  human  mouth,  where  it  maybe  con- 
fused with  the  pneumococcus.  It  has  also  turned  up  un- 
expectedly in  a  variety  of  inflammatory  diseases. 


Micrococcus  Tetragenus  361 


STREPTOCOCCUS  ERYSIPELATIS  (FEHLEISEN). 

The  streptococcus  of  Rosenbach  is  generally  thought  to 
be  identical  with  a  streptococcus  described  by  Fehleisen*  as 
Streptococcus  erysipelatis . 

The  streptococcus  of  erysipelas  can  be  obtained  in  almost 
pure  culture  from  the  serum  which  oozes  from  a  puncture 
made  in  the  margin  of  an  erysipelatous  patch.  They  are 
small  cocci,  usually  forming  chains  of  from  six  to  ten  indi- 
viduals, but  sometimes  reaching  a  hundred  or  more  in  num- 
ber. Occasionally  the  chains  occur  in  tangled  masses. 

They  can  be  cultivated  at  the  room  temperature,  but  grow 
much  better  at  30°  to  37°  C.  They  are  not  particularly  sensi- 
tive to  the  presence  or  absence  of  oxygen,  but  perhaps  de- 
velop a  little  more  rapidly  in  its  presence.  The  cultural 
appearances  are  identical  with  those  of  Streptococcus 
pyogenes. 

When  injected  into  animals  Fehleisen 's  coccus  behaves 
exactly  like  Streptococcus  pyogenes. 


MICROCOCCUS  TETRAGENUS  (GAFFKY). 

General  Characteristics. — Large,  round,  encapsulated  cocci,  regu- 
larly associated  in  groups  of  four,  forming  tetrads.  They  are  non- 
motile,  non-flagellated,  non-sporogenous,  non-liquefying,  non-chromo- 
genic,  non-aerogenic,  aerobic  and  optionally  aerobic,  pathogenic 
for  mice  and  other  small  animals,  and  stain  well  by  all  methods,  in- 
cluding that  of  Gram. 

A  large  micrococcus  grouped  in  fours  and  known  as  Micro- 
coccus  tetragenus  can  sometimes  be  found  in  normal  saliva, 
tuberculous  sputum,  and  more  commonly  in  the  contents  of 
the  cavities  of  tuberculosis  pulmonalis.  It  sometimes  occurs 
in  the  pus  of  acute  abscesses,  and  may  be  of  importance  in 
connection  with  the  pulmonary  abscesses  which  complicate 
tuberculosis.  It  was  discovered  by  Gaffky.f 

Morphology. — The  cocci  are  rather  large,  measuring 
about  i  u  in  diameter.  In  cultures  they  do  not  show  the  regu- 
lar arrangement  in  tetrads  as  constantly  as  in  the  blood  and 
tissues  of  animals,  where  they  occur  in  groups  of  four  sur- 
rounded by  a  transparent  gelatinous  capsule. 

*  "  Verhandlungen  der  Wiirzburger  med.  Gesellschaft,"  1881. 
t  "Archiv.  f.  Chirurgie,"  28,  3. 


362 


Suppuration 


Staining. — The  organisms  stain  well  by  ordinary  methods 
and  beautifully  by  Gram's  method,  by  which  they  can  be 
best  demonstrated  in  tissues. 

Isolation. — The  organism  can  be  isolated  by  inoculating 
a  white  mouse  with  sputum  or  pus  containing  it.  After 
death  it  can  be  recovered  from  the  blood. 

Cultivation. — It  grows  readily  upon  artificial  media. 
Upon  gelatin  plates  small  white  colonies  are  produced  in 


Fig.  108. — Micrococcus  tetragenus  in  spleen  of  infected  mouse. 
(From  Hiss  and  Zinsser  "Text-Book  of  Bacteriology,"  D.  Appleton  & 
Co.,  Publishers.) 

from  twenty-four  to  forty-eight  hours.  Under  the  micro- 
scope they  appear  spheric  or  elongate  (lemon  shaped),  finely 
granular,  and  lobulated  like  a  raspberry  or  mulberry.  When 
superficial  they  are  white  and  elevated,  i  to  2  mm.  in  diam- 
eter. 

Gelatin. — In  gelatin  punctures  a  large  white  surface 
growth  takes  place,  but  development  in  the  puncture  is  very 
scant,  the  small  spheric  colonies  usually  remaining  isolated. 
The  gelatin  is  not  liquefied. 


Micrococcus  Tetragenus  363 

Agar-agar. — Upon  agar-agar  spheric  white  colonies  are 
produced.  They  may  remain  discrete  or  become  confluent. 

Potato. — Upon  potato  a  luxuriant,  thick,  white  growth 
is  formed. 

Blood-serum. — The  growth  upon  blood-serum  is  also 
abundant,  especially  at  the  temperature  of  the  incubator. 
It  has  no  distinctive  peculiarities. 

Pathogenesis. — The  introduction  of  tuberculous  sputum 
or  of  a  minute  quantity  of  a  pure  culture  of  this  coccus  into 
white  mice  usually  causes  a  fatal  bacteremia  in  which  these 


Fig.  109. — Micrococcus  tetragenus;  colony  twenty-four  hours  old  upon 
the  surface  of  an  agar-agar  plate.      X   100  (Heim). 


organisms  are  found  in  small  numbers  in  the  heart's  blood, 
but  are  numerous  in  the  spleen,  lungs,  liver,  and  kidneys. 

Japanese  mice  and  white  mice  are  highly  susceptible  to  the 
organism  and  die  three  or  four  days  after  inoculation. 

House-mice,  field-mice,  and  rabbits  are  comparatively 
immune.  Guinea-pigs  may  die  of  general  septic  infection, 
though  local  abscesses  result  from  subcutaneous  inoculation. 

The  tetracocci,  when  present,  probably  hasten  the  tissue- 
necrosis  in  tuberculous  cavities,  aid  in  the  formation  of  ab- 
scesses of  the  lung,  and  contribute  to  the  production  of  the 
hectic  fever. 

An  interesting  contribution  to  the  relationship  of  this 
coccus  to  human  pathology  has  been  made  by  Lartigau,* 
who  succeeded  in  demonstrating  that  the  tetracoccus  may 

*<<Phila.  Med.  Jour.,"  April  22,  1899. 


364  Suppuration 

be   the  cause  of  a  pseudomembranous  angina,  3  cases  of 
which  came  under  his  observation. 

Bezancon*  has  isolated  this  organism  from  a  case  of  menin- 
gitis. Forneacaf  has  reported  a  case  of  generalized  tetra- 
genous  septicemia. 

BACILLUS  PYOCYANEUS  (GESSARD). 

General  Characteristics. — A  minute,  slender,  actively  motile,  flag- 
ellated, non-sporogenous,  chromogenic  and  feebly  pathogenic,  aerobic 
or  facultative  anaerobic,  liquefying  bacillus,  staining  by  ordinary 
methods,  but  not  by  Gram's  method. 

In  some  cases  pus  has  a  peculiar  bluish  or  greenish  color, 
which  depends  upon  the  presence  of  Bacillus  pyocyaneus 
of  Gessard.l 

Distribution. — The  bacillus  appears  to  be  a  rather  com- 
mon saprophyte,  being  found  in  feces,  manure,  and  water. 


Fig.  no. — Bacillus  pyocyaneus,  from  an  agar-agar  culture.      X  1000 
(Itzerott  and  Niemann). 

It  easily  takes  up  its  residence  upon  the  skin  and  mucous 
membranes,  and  has  been  found  in  the  perspiration.  It 
sometimes  occurs  as  a  saprophyte  upon  the  surgical  dressings 
applied  to  wounds,  and  sometimes  invades  the  tissues 
through  wounds,  to  occasion  dangerous  infections. 

*"Semaine  Medicale,"  1898. 

t"Riforma  Medica,"  1903. 

J  "De  la  Pyocyanine  et  de  son  Microbe,"  These  de  Paris,  1882. 


Bacillus  Pyocyaneus  365 

Morphology. — It  is  a  short,  slender  organism  with  rounded 
ends,  measuring  0.3  x  i  to  2  ^,  according  to  Fliigge;  0.6  x 
2  to  6  /w,  according  to  Ernst,  and  0.6  x  i  ^,  according  to 
Charrin.  It  is  quite  pleomorphous,  which  probably  accounts 
for  the  difference  in  measurements.  It  is  frequently  united 
in  chains  of  four  or  six.  It  is  actively  motile,  has  one 
terminal  flagellum,  and  does  not  form  spores.  It  can  exist 
without  free  oxygen,  though  it  is  an  almost  purely  aerobic 
organism. 

It  closely  resembles  a  harmless  bacillus  found  in  water, 
and  known  as  Bacillus  fluorescens  liquefaciens,  from  which 
Ruzicka*  thinks  it  has  probably  descended 


vi 

Fig.  in. — Bacillus  pyocyaneus.     Colonies  upon  gelatin  (Abbott). 

Staining. — It  stains  well  with  the  ordinary  staining  solu- 
tions, but  not  by  Gram's  method. 

Isolation. — The  isolation  of  the  organism  is  simple,  the 
ordinary  plate  method  being  a  satisfactory  means  of  securing 
it  from  pus  or  other  discharges. 

Cultivation. — Colonies. — The  superficial  colonies  upon 
gelatin  plates  are  small,  irregular,  slightly  greenish,  ill- 
defined,  and  produce  a  distinct  fluorescence  of  the  neigh- 
boring gelatin. 

Microscopic  examination  shows  the  superficial  colonies  to 
be  rounded  and  coarsely  granular,  with  serrated  or  slightly 
filamentous  borders.  They  are  distinctly  green  in  the  center 
and  pale  at  the  edges.  The  colonies  sink  into  the  gelatin  as 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  July  15,  1898,  p.  n. 


366  Suppuration 

the  liquefaction  progresses.  Four  or  five  days  must  elapse 
before  the  medium  is  all  fluid. 

Gelatin  Punctures. — In  gelatin  puncture  cultures  the 
chief  development  of  the  organisms  occurs  at.  the  upper 
part  of  the  tube,  where  a  deep  saucer-shaped  liquefaction 
forms,  slowly  descending  into  the  medium,  and  causing  a 
beautiful  fluorescence.  At  times  a  delicate  scum  forms 
on  the  surface,  sinking  to  the  bottom  as  the  culture  ages, 
and  ultimately  forming  a  slimy  sediment. 

Agar-agar. — Upon  agar-agar  the  growth  developing  all 
along  the  line  of  inoculation  at  first  appears  bright  green. 
The  green  color  depends  upon  a  soluble  pigment  (fluorescein) 
which  soon  saturates  the  culture-medium  and  gives  it  the 
characteristic  fluorescent  appearance.  As  the  culture  ages, 
or  if  the  medium  upon  which  it  grows  contains  much  pep- 
tone, a  second  blue  pigment  (pyocyanin)  develops,  and 
the  bright  green  fades  to  a  deep  blue-green,  dark  blue,  or 
in  some  cases  to  a  deep  reddish-brown  color.  This  pigment 
has  been  made  the  subject  of  a  careful  investigation  by  Jor- 
dan.* Its  formula,  according  to  Ledderhose,  f  is  C14H14N2O. 

A  well-known  feature  of  the  growth  upon  fresh  agar- 
agar,  upon  which  much  stress  has  recently  been  laid  by 
Martin,  J  is  the  formation  of  crystals  in  fresh  cultures. 
Crystal  formation  in  cultures  of  other  bacteria  usually  takes 
place  in  old,  partially  dried  agar-agar  cultures,  but  Bacil- 
lus pyocyaneus  often  produces  crystals  in  a  few  days  upon 
fresh  media.  In  my  experience  freshly  isolated  bacilli  show 
this  power  more  markedly  than  those  which  have  been  for 
some  time  part  of  the  laboratory  stock  of  cultures  and 
frequently  transplanted. 

Bouillon. — In  bouillon  the  organism  produces  a  diffuse 
cloudiness,  a  fluorescence,  and  sometimes  an  indefinite  thin 
pellicle  on  the  surface. 

Potato. — Upon  potato  a  luxuriant  greenish  or  brownish, 
smeary  layer  is  produced. 

Milk. — Milk  is  coagulated  and  peptonized.  It  is  slightly 
acid  for  the  first  day  or  two,  then  becomes  alkaline  again. 

Metabolic  Products. — Apart  from  the  pyocyanin  and 
fluorescin,  the  former  blue,  the  latter  green,  cultures  of  this 
organism  frequently  turn  red  brown.  This  suggested  the 

*  "Journal  of  Experimental  Medicine,"  vol.  iv,  1899. 
t"  Deutsche  Zeitschr.  f.  Chirurgie,"  1888,  Bd.  xxvm. 
t  "Centralbl.  f.  Bakt.,"  xxi,  April  6,  1897,  p.  473. 


Bacillus  Pyocyaneus  367 

formation  of  a  third  pigment,  but  the  work  of  Boland  *  has 
shown  this  to  be  a  transformation  product  of  pyocyanin 
common  in  old  cultures. 

The  organism  produces  a  curdling  ferment,  a  fibrin-  and 
casein-dissolving  ferment,  a  gelatin-dissolving  ferment, 
and  a  bacteriolytic  ferment,  the  pyocyanase  of  Emmerich 
and  Low. 

It  also  produces,  under  favorable  conditions,  a  toxin 
which  has  been  studied  by  Wassermann,  who  found  it  fatal 
in  doses  of  0.2  to  0.5  c.c.  when  intraperitoneally  injected  into 
guinea-pigs.  The  animals  show  peritonitis  and  punctiform 
hemorrhages  on  the  serous  membranes. 

Bullock  and  Hunter  f  found  that  Bacillus  pyocyaneus  also 
produces  a  hemolytic  substance,  pyocyanolysin,  by  which 
corpuscles  of  man,  oxen,  sheep,  apes,  rabbits,  cats,  rats, 
dogs,  and  mice  are  dissolved.  The  peculiar  substance  was 
produced  in  greatest  quantity  in  virulent  cultures  three  or 
four  weeks  old.  JordanJ  believes  that  this  hemolytic 
property  of  the  cultures  depends  solely  upon  the  intense 
alkali  formed  in  old  pyocyaneus  cultures.  Gheorghewski  § 
found  a  leukocyte-destroying  substance  in  the  cultures. 

In  addition  to  the  metabolic  pigments  mentioned,  the  or- 
ganism produces  toxins.  Wassermann  ||  found  that  filtrates 
of  old  cultures  were  more  toxic  for  guinea-pigs  than  the  endo- 
toxins  made  by  lysis  of  dead  bacteria.  The  organism  thus 
produces  both  endo-  and  exotoxins. 

Pathogenesis. — The  bacillus  is  pathogenic  for  the  small 
laboratory  animals,  but  different  cultures  differ  greatly 
in  virulence.  One  c.c.  of  a  virulent  bouillon  culture,  injected 
into  the  subcutaneous  tissue  of  a  guinea-pig  or  a  rabbit, 
caused  rapid  edema,  suppurative  inflammation,  and  death 
in  a  short  time  (twenty-four  hours).  Sometimes  the  animal 
lives  for  a  week  or  more,  then  dies.  There  is  a  marked 
hemorrhagic  subcutaneous  edema  at  the  seat  of  inoculation. 
The  bacilli  can  be  found  in  the  blood  and  in  most  of  the 
tissues.  The  guinea-pig  is  most  susceptible. 

Doses  too  small  to  prove  fatal  sometimes  lead  to  suppu- 
ration, and  the  injection  of  sterilized  cultures  leads  to  simi- 
lar results,  a  relatively  larger  quantity  being  required. 

*  "Centralbl.  f.  Bakt.,"  Bd.  xxv,  1899,  p.  897. 

t  Ibid.,  xxviu,  1900,  p.  865.  |  Ibid.,  Bd.  xxxin,  Ref.  1903. 

§  "Ann.  de  1'Inst.  Pasteur,"  1899,  xin. 

||  "Zeitschrift  fur  Hygiene,  1896,  xxn. 


368  Suppuration 

Intraperitoneal  injections  cause  purulent  peritonitis. 

Blum*  reports  a  case  of  pyocyaneus  infection  with  endo- 
carditis in  a  child. 

Lartigau,f  in  his  study  of  "  The  Bacillus  Pyocyaneus  as 
a  Factor  in  Human  Pathology,"  sums  up  what  is  known 
about  this  role  of  the  organism  as  follows:  "  The  Bacillus 
pyocyaneus,  like  many  pathogenic  micro-organisms,  is 
occasionally  found  in  a  purely  saprophytic  role  in  various 
situations  in  the  human  economy.  It  has  been  found  in  the 
saliva  by  Pansini,  in  sputum  by  Frisch,  and  in  the  sweat  by 
Kberth  and  Audanard.  Abelous  demonstrated  its  presence 
in  the  stomach  as  a  saprophyte.  Its  existence  in  suppurat- 
ing wounds  has  long  been  known,  and  Koch  early  detected 
its  presence  in  tuberculous  cavities,  regarding  it  as  an 
organism  incapable  of  playing  any  pathologic  role.  The 
etiologic  relation  of  the  organism  to  certain  cases  of  purulent 
otitis  media  in  children  was  pointed  out  by  Martha,  Mag- 
giora  and  Gradenigo,  Babes,  Kossel,  and  others.  H.  C. 
Ernst  obtained  it  from  a  pericardial  exudate  during  life.  G. 
Blumer  demonstrated  its  presence  in  practically  pure  cul- 
tures in  a  case  of  acute  angina  simulating  diphtheria; 
Jadkewitsch,  B.  Motz,  and  Le  Noir  obtained  the  bacillus 
in  cases  of  urinary  infection.  The  cases  of  Triboulet, 
Karlinski,  Oettinger,  Bhlers,  and  Barker  are  interesting 
instances  of  its  role  in  cutaneous  lesions. 

"  In  addition  to  these  lesions,  other  morbid  processes 
have  been  associated  in  some  cases  with  the  bacillus  of 
blue  pus,  such  as  meningitis  and  bronchopneumonia,  by 
Monnier;  diarrhea  of  infants,  by  Neumann,  Williams,  Thier- 
celin  and  Lesage,  and  other  observers;  dysentery,  by  Cal- 
mette  and  by  Lartigau;  and  general  infection,  by  Khlers, 
Neumann,  Oettinger,  Karlinski,  Monnier,  Krannhals,  Cal- 
mette,  Finkelstein,  and  I,.  F.  Barker." 

Nine  additional  cases  of  human  infection  are  reported  by 
Perkins.  { 

Immunity. — Immunity  against  pyocyaneus  infection  de- 
velops after  a  few  inoculations  with  attenuated  or  sterilized 
cultures.  These  are  easily  prepared,  the  thermal  death-point 
determined  by  Sternberg  being  56°  C.  It  also  follows  in- 
jection of  either  the  endotoxin  or  the  exotoxin.  In  the 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Feb.  10,  1899,  xxv,  No.  4. 

t  "Phila.  Med.  Jour.,"  Sept.  17,  1898. 

t  "Jour,  of  Med.  Research,"  vol.  vi,  1901,  p.  281. 


Bacillus  Proteus  Vulgaris  369 

immunity  resulting  from  the  treatment  with  killed  cultures 
the  serum  of  the  animal  becomes  agglutinative  and  bac- 
tericidal ;  in  the  immunity  resulting  from  treatment  with  the 
exotoxin,  antitoxin  is  produced. 

BACILLUS  PROTEUS  VULGARIS  (HA USER). 

General  Characteristics. — An  actively  motile,  flagellated,  non- 
sporogenous,  non-chromogenic,  liquefying,  aerobic  and  optionally 
anaerobic,  doubtfully  pathogenic,  aerogenic  bacillus,  easily  cultivated 
on  artificial  media  and  readily  stained  by  the  ordinary  methods,  though 
not  by  Gram's  method. 

This  bacillus  was  first  found  by  Hauser*  in  decomposing 
animal  infusions,  usually  in  company  with  two  closely  allied 
forms,  Proteus  mirabilis  and  Proteus  zenkeri,  which,  as  the 
experiments  and  observations  of  Sanfelice  and  others  show, 
may  be  identical  with  it.  According  to  Kruse,  it  is  quite 
probable  that  the  mixed  species  formerly  called  Bacterium 
termo  was  largely  made  up  of  the  proteus. 

Distribution. — The  organism  is  a  common  saprophyte 
and  is  very  abundant  in  water,  earth,  and  air.  It  is  to  be 
expected  wherever  putrefactive  change  is  in  progress.  It  is 
a  common  mistake  for  the  novice  to  look  upon  it  as  a  member 
of  the  Bacillus  coli  group. 

Morphology. — The  bacilli  are  variable  in  size  and  shape — 
pleomorphic — and  are  named  proteus  from  this  peculiarity. 
Some  differ  very  little  from  cocci,  some  are  more  like  the 
colon  bacillus  in  shape,  others  form  long  filaments,  and  oc- 
casional spirulina  forms  are  met  with.  True  spirals  are 
never  found.  All  of  the  forms  mentioned  may  be  found  in 
pure  cultures  of  the  same  organism.  The  diameter  of  the 
bacillus  is  usually  about  0.6  /w,  but  the  length  varies  from  1.2 
fi  or  less  to  4  f*  or  more.  No  spores  are  formed.  The  organ- 
isms are  actively  motile.  The  long  filaments  frequently  form 
loops  and  tangles.  Flagella  are  present  in  large  numbers. 
Upon  one  of  the  long  bacilli  as  many  as  one  hundred  have 
been  counted.  Involution  forms  are  frequent  in  old  cultures. 

Staining. — The  bacilli  stain  well  by  the  ordinary  methods. 
Gram's  method  usually  fails. 

Cultivation. — The  proteus  is  easily  cultivated  and  grows 
well  in  all  the  artificial  media. 

Colonies. — Upon  gelatin  plates  a  typical  phenomenon  is 

*"Ueber  Faulnissbakterien,"  Leipzig,  1885. 
24 


370 


Suppuration 


observed  in  connection  with  the  development  of  the  colonies, 
for  the  most  advantageous  observation  of  which  the  medium 
used  for  making  the  cultures  should  contain  5  instead  of  10 
per  cent,  of  gelatin.  Kruse  *  describes  the  phenomenon  as 
follows:  "At  the  temperature  of  the  room,  rounded,  saucer- 
shaped  depressions,  with  a  whitish  central  mass  surrounded 
by  a  lighter  zone,  are  quickly  formed.  Under  low  magni- 
fication the  center  of  each  is  seen  to  be  surrounded  by 
radiations  extending  in  all  directions  into  the  solid  gelatin, 
and  made  up  of  chains  of  bacilli.  Between  the  radiations 


Fig.  112. — Swarming  islands  of  proteus  bacilli  on  the  surface  of  gelatin; 
X  650  (Hauser). 

and  the  granular  center  bacteria  are  seen  in  active  motion. 
Upon  the  surface  the  colony  extends  as  a  thin  patch,  con- 
sisting of  a  layer  of  bacilli  arranged  in  threads,  sending 
numerous  projections  from  the  periphery.  Under  certain 
conditions  the  wandering  of  the  processes  can  be  directly 
observed  under  the  microscope.  It  depends  not  only  upon 
the  culture-medium,  but,  in  part,  upon  the  culture  itself. 
Entire  groups  of  bacilli  or  single  threads,  by  gradual  exten- 
sion and  circular  movement,  detach  themselves  from  the 
colony  and  wander  about  upon  the  plate.  From  the  radi- 

*  Flugge's  "  Die  Mikroorganismen." 


Bacillus  Proteus  Vulgaris  371 

ated  central  part  of  the  colony  peculiar  zooglea  are  formed, 
having  a  sausage  or  screw  shape,  or  wound  in  spirals  like 
a  corkscrew.  The  younger  colonies,  which  have  not  yet 
reached  the  surface  of  the  gelatin,  are  more  compact,  rounded 
or  nodular,  later  covered  with  hair-like  projections,  and 
becoming  radiated  like  the  superficial  colonies." 

If  the  culture-medium  be  concentrated,  or  the  culture 
has  been  frequently  transplanted,  the  phenomenon  is  less 
marked  and  may  not  occur. 

Bouillon. — In  this  medium  the  organism  grows  rapidly, 
and  quickly  clouds  the  fluid.  A  pellicle  soon  forms  upon  the 
surface  and  a  mucilaginous  sediment  occurs  later. 

Gelatin  Punctures. — Puncture  cultures  in  gelatin  are  not 
characteristic.  A  stocking-like  liquefaction  of  the  gelatin 
extends  so  rapidly  that  the  entire  gelatin  is  liquefied  in  a  few 
days.  Anaerobic  cultures  do  not  liquefy. 

Agar-agar. — Upon  agar-agar  the  bacillus  forms  a  moist, 
thin,  transparent,  rapidly  extending  layer  which  rarely 
reaches  the  sides  of  the  tube.  Upon  agar-agar  plates  ame- 
boid movement  of  the  colonies  may  also  occur. 

Potato. — Upon  potato  the  growth  occurs  in  the  form  of 
a  smeary  patch  of  soiled  appearance. 

Milk  is  coagulated. 

Metabolic  Products. — The  bacillus  usually  produces 
alkalies.  Indol  and  phenol  are  formed  from  the  peptone  of 
the  culture-media.  Nitrates  are  reduced  to  nitrites,  and 
then  partly  reduced  to  ammonia.  In  most  culture-media 
not  containing  sugar  the  bacillus  produces  a  very  disagree- 
able odor. 

In  culture-media  containing  either  grape-  or  cane-sugar 
fermentation  occurs  both  in  the  presence  and  in  the  absence 
of  oxygen.  Milk-sugar  is  not  decomposed. 

Pathogenesis. — It  is  a  question  whether  or  not  Bacillus 
proteus  is  to  be  ranked  among  the  pathogenic  bacteria. 
Small  doses  are  harmless  for  the  laboratory  animals;  large 
doses  produce  abscesses.  A  toxic  substance  resulting  from 
the  metabolism  of  the  organism  seems  to  be  the  cause  of 
death  when  considerable  quantities  of  a  culture  are  injected 
into  the  peritoneal  cavity  or  blood-vessels.  The  bacilli  do 
not  seem  able  to  multiply  in  the  healthy  animal  body,  but 
can  do  so  when  previous  disease  or  injury  of  its  tissues  has 
taken  place. 

The  proteus  has  been  secured  in  cultures  from  wound  and 


372  Suppuration 

puerperal  infections,  purulent  peritonitis,  endometritis,  and 
pleurisy.  When  the  local  lesion  is  limited,  as  in  endometritis, 
the  danger  of  toxemia  is  slight  ;  but  when  widespread,  as  the 
peritoneum,  it  may  prove  serious.  Bacillus  proteus  has  also 
been  found  in  acute  infectious  jaundice  and  in  acute  febrile 
icterus,  or  Weil's  disease. 

Bordoni-Uffredizzi  has  shown  that  the  proteus  quite 
regularly  invades  the  tissues  after  death,  though  it  ap- 
pears unable  to  maintain  an  independent  existence  in  the 
tissues  during  life,  and  is  probably  of  importance  only  when 
present  in  association  with  other  bacteria.  It  at  times 
grows  abundantly  in  the  urine,  and  may  produce  primary 
inflammation  of  the  bladder.  The  inflammatory  process 
may  also  extend  from  the  bladder  to  the  kidney,  and  so 
prove  quite  serious. 

Epidemics  of  meat-poisoning  have  been  thought  to  depend 
upon  Bacillus  proteus.  One  of  them  was  studied  by  Wesen- 
berg,  *  who  cultivated  the  organism  from  the  putrid  meat  by 
which  63  persons  were  made  ill.  Sil  verschmidt  f  and  PfuhlJ 
have  made  similar  investigations  with  similar  results. 


AND  SUPPURATION. 

The  process  of  suppuration  is  not  confined  to  bacterial 
micro-organisms,  but  is  shared  to  a  limited  extent  by  the 
protozoa.  Thus,  Bntamoeba  histolytica  (q.  v.)  is,  to  all 
appearances,  the  sole  excitant  of  the  abscesses  of  the  liver 
secondary  to  dysentery.  It  is  true  that  these  are  cold 
abscesses  and  necrotic  rather  than  distinctly  purulent  in 
character,  yet  it  seems  best  to  speak  of  the  organism  in  this 
connection. 

Bntamoeba  buccalis  (Prowazek§)  is  a  small  ameba  that 
has  been  found  in  purulent  exudates  in  the  oral  tissues  of 
persons  with  carious  teeth. 

Amoeba  kartulisi  (Doflein||)  appears  to  be  capable  of  ex- 
citing suppuration.  It  was  found  by  Kartulis  in  the  pus 
from  an  abscess  of  the  right  side  of  the  lower  jaw.  The 
patient  was  a  man  aged  forty-three  years  who  had  been 

*  "Zeitschrift  fur  Hygiene,"  etc.,  1898,  xxvm. 

t  Ibid.,  1899,  xxx. 

J  Ibid.,  1900,  xxxv. 

§  "Arbeitena.  d.  Kaiserl.  Gesundh.-Amt.,"  xxi,  i,  Bull.  1904,  p.  42. 

||  "Die  Protozoa  als  Krankheitserreger,"  Jena.,  1901,  p.  30. 


Miscellaneous  Organisms  Described  Elsewhere    373 

operated  upon  for  the  removal  of  a  piece  of  bone.  It  is  30 
to  38  {*.  in  diameter,  is  actively  motile.  Its  coarse  protoplasm 
contains  red  and  white  blood-corpuscles.  Kartulis*  found 
the  same  organism  five  times  in  other  cases,  and  Flexnerf 
found  it  also. 

Amoeba  mortinatalium,  described  by  Smith  and  Weidman,| 
was  found  in  distributed  small  purulent  foci  in  the  kidneys 
and  other  organs  of  a  still-born  fetus. 

MISCELLANEOUS  ORGANISMS  OF  SUPPURATION  DESCRIBED 
MORE  FULLY  ELSEWHERE. 

Before  leaving  the  subject,  attention  must  be  directed 
to  other  bacteria  that  under  exceptional  circumstances 
become  the  cause  of  suppuration.  Among  these  are  the 
pneumococcus  of  Frankel  and  Weichselbaum,  the  typhoid 
bacillus,  and  the  Bacillus  coli  communis.  These  organisms 
are  considered  under  separate  and  appropriate  headings,  to 
which  the  reader  is  advised  to  refer. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk."  1903,  xxxni,  p.  471. 

t  "Bulletin  of  the  Johns  Hopkins  Hospital,"  1892,  xxv. 

I  "University  of  Pennsylvania  Medical  Bulletin,"  Sept.,  1910. 


CHAPTER   II. 
MALIGNANT  EDEMA. 

BACILLUS  (EDEMATIS  MALIGNI  (KOCH). 

General  Characteristics. — A  motile,  flagellated,  sporogenous,  an- 
aerobic, liquefying,  aerogenic,  non-chromogenic,  pathogenic  bacillus  of 
the  soil,  readily  stained  by  the  ordinary  methods,  but  not  by  Gram's 
method. 

This  organism  was  originally  found  by  Pasteur*  in  putres- 
cent  animal  infusions  and  called  -by  him  (1875)  Vibrion 
septique.  It  was  later  more  carefully  studied  and  described 
by  Koch.f 

Distribution. — The  organism  is  widely  distributed  in 
nature,  being  commonly  present  in  garden  earth.  It  is  also 
found  in  dust,  in  waste  water  from  houses,  and  sometimes  in 
the  intestinal  canals  of  animals. 

Morphology. — The  bacillus  of  malignant  edema  is  a  large 
rod-shaped  organism  with  rounded  ends,  measuring  2  to  10  /w 
by  0.8  to  i.o  [i.  It  is  usually  motile,  and  possesses  many 
flagella.  It  produces  oval  endospores  centrally  situated  and 
giving  a  barrel  shape  to  the  parent  bacillus. 

Staining. — The  bacillus  stains  well  with  ordinary  cold 
aqueous  solutions  of  the  anilin  dyes,  but  not  by  Gram's 
method. 

Cultivation. — The  organism  is  a  strict  anaerobe,  but  under 
conditions  by  which  provision  is  made  for  the  removal  of 
oxygen,  grows  well  both  at  the  room  temperature  and  at  that 
of  the  incubator.  It  is  not  difficult  to  secure  in  pure  culture, 
being  most  easily  obtained  from  the  edematous  tissues  of 
guinea-pigs  and  rabbits  inoculated  with  garden  earth. 

The  colonies  which  develop  upon  the  surface  of  gelatin 
kept  under  anaerobic  conditions  appear  to  the  naked  eye 
as  small  shining  bodies  with  liquid,  grayish-white  contents. 
Under  the  microscope  they  appear  filled  with  a  tangled 
mass  of  long  filaments  which  under  a  high  power  exhibit 

*  "Bull.  Acad.  Med.,"  1877  and  1881. 

t  "Mittheilungen  aus  dem  kaiserl.  Gesundheitsamte,"  i,  53. 
374 


Metabolic  Products 


375 


active  movement.  The  edges  of  the  colony  have  a  fringed 
appearance,  much  like  the  colonies  of  the  hay  or  potato  ba- 
cillus. 

In  gelatin  and  agar-agar  tube  cultures  the  characteristic 
growth  cannot  be  observed  in  a  puncture,  because  of  the  air 
which  remains  in  the  path  of  the  wire,  unless  the  tube  be 
placed  under  anaerobic  conditions.  The  best  preparation, 
therefore,  is  made  by  heating  the  gelatin  to  expel  any  air  it 
may  contain,  inoculating  it  while  still  liquid,  and  solidifying 
it  in  cold  (iced)  water.  In  such  a  tube  the  bacilli  develop 
in  globular  circumscribed  areas  of  cloudy  liquefaction 


:  •> 


Fig.  1 13. — Bacillus  of  malignant  edema,  from  the  body- juice  of  a  guinea- 
pig  inoculated  with  garden  earth.      X  1000  (Frankel  and  Pfeiffer). 

which  contain  a  small  amount  of  gas.  In  gelatin  to  which  a 
little  grape-sugar  has  been  added  the  gas  production  is 
marked.  The  gas  is  partly  inflammable,  partly  not.  A  dis- 
tinct odor  accompanies  the  gas  production,  and  is  especially 
noticeable  in  agar-agar  cultures.  In  bouillon  diffuse  cloud- 
ing occurs,  followed  by  the  formation  of  a  sediment.  No 
surface  growth  occurs.  Milk  is  slowly  coagulated.  It  grows 
well  upon  the  surface  of  potato  and  blood-serum  under  con- 
ditions of  strict  anaerobiosis. 

Metabolic  Products. — Of  the  toxic  products  of  the  organ- 
ism nothing  definite  is  known.  It  decomposes  albumin,  form- 
ing fatty  acids,  leucin,  hydroparacumaric  acid,  and  an  oil 


376 


Malignant  Edema 


with  an  offensive  odor.  Among  the  gases  formed,  carbonic 
acid,  hydrogen,  and  marsh  gas  have  been  detected. 

Pathogenesis. — When  introduced  beneath  the  skin,  the 
bacillus  is  pathogenic  for  a  large  number  of  animals — mice, 
guinea-pigs,  rabbits,  horses,  dogs,  sheep,  goats,  pigs,  calves, 
chickens,  and  pigeons.  Cattle  seem  to  be  immune. 

Giinther  points  out  that  the  simple  inoculation  of  the 
bacillus  upon  an  abraded  surface  is  insufficient  to  produce 
infection,  because  the  presence  of  oxygen  is  detrimental  to 
its  growth.  When  the  bacilli  are  deeply  introduced  beneath 
the  skin,  infection  occurs. 

Mice,  guinea-pigs,  and  rabbits  sicken  and  die  in  about 
forty-eight  hours. 


Fig.  114. — Bacillus  cedematis,  dextrose  gelatin  culture  (Giinther). 

Lesions. — In  the  blood  the  bacilli  are  few  because  of  the 
loosely  combined  oxygen  it  contains.  The  great  majority 
of  the  bacilli  occupy  the  subcutaneous  tissue,  where  very 
little  oxygen  is  present  and  the  conditions  of  growth  are 
good.  The  autopsy  shows  a  marked  subcutaneous  edema 
containing  immense  numbers  of  the  bacilli.  If  the  animal 
be  permitted  to  remain  undisturbed  for  some  time  after 
death,  the  bacilli  spread  to  the  circulatory  system  and  reach 
all  the  organs. 

Brieger  and  Ehrlich*  have  reported  2  cases  of  malignant 
edema  in  man.  Both  occurred  in  typhoid  fever  patients 
subcutaneously  injected  with  musk,  the  infection  no  doubt 
resulting  from  impurities  in  the  therapeutic  agent. 

*  "Berliner  klin.  Wochenschrift,"  1882,  No.  44. 


I 


Immunity  377 

Grigorjeff  and  Ukke*  have  observed  another  interesting 
case  of  typhoid  fever  with  intestinal  ulcerations,  through 
which  infection  by  the  bacillus  of  malignant  edema  took  place. 
The  case  was  characterized  by  interstitial  emphysema  of 
the  subcutaneous  tissue  of  the  neck  and  breast,  gas  bubbles 
in  the  muscles,  and  a  transformation  of  the  entire  liver  into 
a  spongy  porous  mass  of  a  grayish-brown  color.  The  spleen  was 
enlarged  and  soft,  and  contained  a  few  gas-bubbles.  Though 
the  writers  consider  this  organism  to  be  the  bacillus  of  malig- 
nant edema,  the  general  impression  one  receives  from  the 
description  of  the  lesions  suggests  that  it  was  Welch's  Bacillus 
aerogenes  capsulatus. 

No  case  is  reported  in  which  healthy  men  have  been  in- 
fected with  malignant  edema. 

Immunity. — Cornevin  found  that  the  passage  of  the  bacil- 
lus through  white  rats  diminished  its  virulence,  and  that  the 
animals  of  various  species  that  recovered  were  immune 
against  the  virulent  organisms.  Roux  and  Chamberlandf 
found  that  the  filtered  cultures  were  toxic  and  that  animals 
could  be  immunized  by  injection  with  this  toxic  filtrate. 


GASEOUS  EDEMA. 

BACILLUS  AEROGENES  CAPSULATUS  (WELCH). 

General  Characteristics. — A  large,  stout,  non-motile,  non-flagel- 
lated, sporogenous,  non-chromogenic,  purely  anaerobic,  markedly  aero- 
genie,  doubtfully  pathogenic  bacillus,  easily  cultivated  in  artificial 
media,  readily  stained  by  the  ordinary  methods  and  by  Gram's  method. 

This  disease  is  caused  by  an  interesting  micro-organism 
described  by  Welch,  and  subsequently  studied  by  Welch 
and  Nuttall,  {  Welch  and  Flexner,,§  and  others.  Welch  said 
at  the  meeting  of  the  Society  of  American  Bacteriologists  held 
at  Philadelphia,  December  30,  1904,  that  he  believed  this  or- 
ganism to  be  identical  with  Kline's  Bacillus  enteritidis  sporo- 
genes,  and  that  it  belongs  to  the  butyric  acid  group.  It  is 
probably  also  identical  with  Bacillus  phlegmone  emphysema- 
tose  of  Frankel.il  It  was  first  secured  by  Welch  from  the 

*  "  Militar-medizin.  Jour.,"  1898,  p.  323. 
t  "Ann.  de  1'Inst.  Pasteur,"  1887. 

f'Bull.  of  the  Johns  Hopkins  Hospital,"  July  and  Aug.,  1892, 
vol.  viii,  No.  24. 

§  "Jour,  of  Experimental  Medicine,"  Jan.,  1896,  vol.  i,  No.  i,  p.  6. 
||  "Centralbl.  f.  Bakt.,"  etc.,  Bd.  xm,  p.  13. 


378  Gaseous  Edema 

body  of  a  man  dying  suddenly  of  aortic  aneurysm  with  a  pe- 
culiar gaseous  emphysema  of  the  subcutaneous  tissues  and  in- 
ternal organs,  and  a  copious  formation  of  gas  in  the  blood- 
vessels. The  blood  was  thin  and  watery,  of  a  lac  color,  and 
contained  many  large  and  small  gas  bubbles,  and  many 
bacilli,  which  were  also  obtained  from  it  and  the  various 
organs,  especially  in  the  neighborhood  of  the  gas  bubbles, 
in  nearly  pure  culture.  The  coloring-matter  of  the  blood 
was  dissolved  out  of  the  corpuscles  and  stained  the  tissues 
a  deep  red. 

Distribution. — The  organism  is  apparently  of  wide  dis- 
tribution.    It  is  believed  that  the  natural  habitat  of  the 


Fig.  115. — Bacillus  aerogenes  capsulatus  (from  photograph  by  Prof. 
Simon  Flexner). 

bacillus  is  the  soil,  but  there  is  reason  to  think  that  it  com- 
monly occurs  in  the  intestine,  and  may  occasionally  be  found 
upon  the  skin. 

Morphology. — The  bacillus  is  a  large  organism,  measuring 
3-5  p.  in  length,  about  the  thickness  of  the  anthrax  bacillus, 
with  ends  slightly  rounded,  or,  when  joined,  square  (Fig. 
115).  It  occurs  chiefly  in  pairs  and  in  irregular  groups,  but 
not  in  chains,  in  this  particular  differing  from  the  anthrax 
bacillus.  In  culture  media  it  is  usually  straight,  with  slightly 
rounded  ends.  In  old  cultures  the  rods  may  be  slightly 
bent,  and  involution  forms  occur.  The  bacillus  varies  some- 
what in  size,  especially  in  length,  in  different  culture-media. 


Staining  379 

It  usually  appears  thicker  and  more  variable  in  length  in 
artificial  cultures  than  in  the  blood  of  animals. 

The  bacillus  is  not  motile  and  has  no  flagella.  Hndo- 
spores  are  formed  upon  Loffler's  blood-serum. 

It  was  at  first  thought  that  the  bacillus  produced  no  spores, 
but  Dunham*  found  that  spores  were  produced  upon  blood- 
serum,  and  especially  upon  Loffler's  blood-serum  bouillon 
mixture.  The  spores  resist  desiccation  and  exposure  to  the 
air  for  ten  months.  They  stain  readily  in  hot  solutions  of 
fuchsin  in  anilin  water,  and  are  not  decolorized  by  a  mod- 
erate exposure  to  the  action  of  3  per  cent,  solution  of 
hydrochloric  acid  in  absolute  alcohol.  They  are  oval,  and 
are  usually  situated  near  the  middle  of  the  bacillus,  which 
is  distended  because  of  the  large  size  of  the  spore  and  bulges 
at  the  sides. 

Staining. — The  organism  stains  well  with  the  ordinary 
stains,  and  retains  the  color  well  in  Gram's  method.  When 
stained  with  methylene-blue  a  granular  or  vacuolated  ap- 
pearance is  sometimes  observed,  due  to  the  presence  of  un- 
stained dots  in  the  cytoplasm. 

Usually  in  the  body-fluids  and  often  in  cultures  the  bacilli 
are  surrounded  by  distinct  capsules — clear,  unstained  zones. 
To  demonstrate  this  capsule  to  the  best  advantage,  Welch 
and  Nuttall  devised  the  following  special  stain: 

A  cover  is  thinly  spread  with  the  bacilli,  dried,  and  fixed 
without  overheating.  Upon  the  surface  prepared,  glacial 
acetic  acid  is  dropped  for  a  few  moments,  then  allowed  to 
drain  off,  and  at  once  replaced  by  a  strong  aqueous  solution 
of  gentian  violet,  which  is  poured  off  and  renewed  several 
times  until  the  acid  has  been  replaced  by  the  stain.  The 
specimen  is  then  examined  in  the  coloring  solution,  after 
soaking  up  the  excess  with  filter-paper,  the  thin  layer  of 
coloring  fluid  not  interfering  with  a  clear  view  of  the  bac- 
teria and  their  capsules.  After  mounting  in  Canada  balsam 
the  capsules  are  not  nearly  so  distinct.  The  width  of  the 
capsule  varies  from  one-half  to  twice  the  thickness  of  the 
bacillus.  Its  outer  margin  is  stained,  leaving  a  clear  zone 
immediately  about  the  bacillus. 

The  bacillus  is  anaerobic  and  aerogenic.  It  grows  upon 
all  culture-media  at  the  room  temperature,  though  better 
at  the  temperature  of  incubation. 

*  "Bull,  of  the  Johns  Hopkins  Hospital,"  April,  1897,  p.  68. 


38o 


Gaseous  Edema 


r 


Cultivation. — Gelatin. — It  grows  in  ordinary  neutral  or 
alkaline  gelatin,  but  better  in  gelatin 
containing  glucose,  in  which  the  char- 
acteristic gas  production  is  marked. 
Soft  media,  made  with  5  instead  ot 
10  per  cent,  of  the  crude  gelatin,  is 
said  to  be  better  than  the  standard 
preparation. 

There  is  no  distinct  liquefaction  of 
the  medium,  but  in  5  per  cent,  gelatin 
softening  can  sometimes  be  demon- 
strated by  tilting  the  tube  and  observ- 
ing that  the  gas  bubbles  change  their 
position,  as  well  as  by  noticing  that 
the  growth  tends  to  sediment. 

Agar-agar. — In  making  agar-agar 
cultures  careful  anaerobic  precautions 
must  be  observed.  The  tubes  should 
contain  considerably  more  than  the 
usual  quantity  of  the  medium,  which 
should  be  boiled  and  freshly  solidified 
before  using.  The  implantation  should 
be  deeply  made  with  a  long  wire.  The 
growth  takes  place  slowly  unless  such 
tubes  are  placed  in  a  Buchner's  jar  or 
other  anaerobic  device.  The  deeper 
colonies  are  the  largest.  Sometimes 
the  growth  only  takes  place  within 
10— 12  mm.  of  the  surface;  at  others, 
within  3-4  cm.  of  it.  After  repeated 
cultivation  the  organisms  seem  to 
become  accustomed  to  the  presence 
of  oxygen,  and  will  grow  higher  up  in 
the  tube  than  when  freshly  isolated. 

Colonies. — The  colonies  seen  in  the 
culture-media  are  grayish-white  or 
brownish- white  by  transmitted  light, 
and  sometimes  exhibit  a  central  dark 
dot.  At  the  end  of  twenty-four  hours 
the  larger  colonies  do  not  exceed  0.5- 
i.o  mm.  in  diameter,  though  they 
may  subsequently  attain  a  diameter 
of  2—3  mm.  or  more.  Their  first  appearance  is  as  little 


Fig.  1 1 6.  —  Bacillus 
aerogenes  capsulatus, 
with  gas  production 
(from  photograph  by 
Prof.  Simon  Flexner). 


Cultivation  381 

spheres  or  ovals,  more  or  less  flattened,  with  irregular  con- 
tours, due  to  the  presence  of  small  projecting  prongs,  which 
are  quite  distinct  under  a  lens.  The  colonies  may  appear  as 
little  irregular  masses  with  projections. 

After  several  days  or  weeks,  single,  well-shaped  colonies 
may  attain  a  large  size  and  be  surrounded  by  projections, 
either  in  the  form  of  little  knobs  or  spikes  or  of  fine  branch- 
ings— hair-like  or  feathery.  Their  appearance  has  been 
compared  to  thistle-balls  or  powder-puffs  and  to  thorn-apples. 
When  the  growth  takes  place  in  the  puncture,  the  feathery 
projections  are  continuous.  Bubbles  of  gas  make  their  ap- 
pearance in  plain  agar  as  well  as  in  sugar-agar,  though,  of 
course,  less  plentifully.  They  first  appear  in  the  line  of 
growth;  afterward  throughout  the  agar,  often  at  a  distance 
from  the  actual  growth.  Any  fluid  collecting  about  the  bub- 
bles or  at  the  surface  of  the  agar-agar  may  be  turbid  from 
the  presence  of  bacilli.  The  gas-production  is  more  abun- 
dant at  37°  C.  than  at  the  room  temperature. 

The  agar-agar  is  not  liquefied  by  the  growth  of  the  bacillus, 
but  is  often  broken  up  into  fragments  and  forced  into  the 
upper  part  of  the  tube  by  the  excessive  gas-production. 

In  its  growth  the  bacillus  produces  considerable   acid. 

Bouillon. — In  bouillon  growth  does  not  occur  in  tubes 
exposed  to  the  air,  but  when  the  tubes  are  placed  in  Buchner's 
jars,  or  kept  under  anaerobic  conditions,  it  occurs  with  abun- 
dant gas-formation,  especially  in  glucose-bouillon,  with  the 
formation  of  a  frothy  layer  on  the  surface.  The  growth  is 
rapid  in  development,  the  bouillon  becoming  clouded  in  two 
to  three  hours.  After  a  few  days  the  bacilli  sediment  and 
the  bouillon  again  becomes  clear.  The  reaction  of  the  bouil- 
lon becomes  strongly  acid. 

Milk. — In  milk  the  growth  is  rapid  and  luxuriant  under 
anaerobic  conditions,  but  does  not  take  place  in  cultures 
exposed  to  the  air.  The  milk  is  coagulated  in  from  twenty- 
four  to  forty-eight  hours,  the  coagulum  being  either  uniform 
or  firm,  retracted,  and  furrowed  by  gas  bubbles.  When 
litmus  has  been  added  to  the  milk,  it  becomes  decolorized 
when  the  culture  is  kept  without  oxygen,  but  turns  pink 
when  it  is  exposed  to  the  air. 

Potato. — The  bacillus  will  also  grow  upon  potato  when  the 
tubes  are  inclosed  in  an  anaerobic  apparatus.  There  is  a 
copious  gas-development  in  the  fluid  at  the  bottom  and  sides 
of  the  tube,  so  that  the  potato  becomes  surrounded  by  a 


382  Gaseous  Edema 

froth.  After  complete  absorption  of  the  oxygen  a  thin, 
moist,  grayish-white  growth  takes  place  upon  the  surface  of 
the  medium. 

Vital  Resistance. — The  vital  resistance  of  the  organism 
is  not  great.  Its  thermal  death-point  was  found  to  be  58° 
C.  after  ten  minutes'  exposure.  Cultures  made  by  displacing 
the  air  with  hydrogen  are  less  vigorous  than  those  in  which 
the  oxygen  is  absorbed  from  the  air  by  pyrogallic  acid.  It 
was  found  that  in  the  former  class  of  cultures  the  bacillus 
died  in  three  days,  while  in  the  absorption  experiments  it 
was  kept  alive  at  the  body  temperature  for  one  hundred  and 
twenty-three  days.  It  is  said  to  live  longer  in  plain  agar 
than  in  sugar-agar.  To  keep  the  cultures  alive  it  has  been 
recommended  to  seal  the  agar-agar  tube  after  two  or  three 
days'  growth. 

Pathogenesis. — The  pathogenic  powers  of  the  bacillus  are 
limited,  and  while  in  some  infected  cases  it  seems  to  be  the 
cause  of  death,  its  power  to  do  mischief  in  the  body  seems  to 
depend  entirely  upon  the  pre-existence  of  depressing  and 
devitalizing  conditions  predisposing  to  its  growth. 

Being  anaerobic,  the  bacilli  are  unable  to  live  in  the  cir- 
culating blood,  though  they  grow  in  old  clots  and  in  cavities, 
such  as  the  uterus,  etc.,  where  little  oxygen  enters,  and  from 
which  they  enter  the  blood  and  are  distributed. 

In  support  of  these  views  Welch  and  Nuttall  show  that 
when  a  healthy  rabbit  is  injected  with  2.5  c.c.  of  a  fresh 
sugar-bouillon  into  the  ear-vein,  it  usually  recovers  without 
any  evident  symptoms.  After  similar  injection  with  but  i 
c.c.  of  the  culture,  a  pregnant  rabbit  carrying  two  dead 
embryos,  died  in  twenty-one  hours.  It  seems  that  the 
bacilli  were  first  able  to  secure  a  foothold  in  the  dead  em- 
bryos, and  there  multiplied  sufficiently  to  bring  about  the 
subsequent  death  of  the  mother. 

After  death,  when  the  blood  is  no  longer  oxygenated,  the 
bacilli  grow  rapidly,  with  marked  gas-production,  which  in 
some  cases  is  said  to  cause  the  body  to  swell  to  twice  its 
natural  size.  The  effect  upon  guinea-pigs  does  not  differ 
from  that  upon  rabbits,  though  gaseous  phlegmons  are 
sometimes  produced. 

Pigeons,  when  subcutaneously  inoculated  in  the  pectoral 
region,  frequently  succumb.  Following  the  injection  gas- 
production  causes  the  tissues  of  the  chest  to  become  emphy- 


Pathogenesis  383 

sematous.  The  birds  usually  die  in  from  seven  to  twenty- 
four  hours,  but  may  recover. 

Intraperitoneal  inoculation  of  animals  sometimes  causes 
fatal  purulent  peritonitis. 

Sources  of  Infection. — The  infection  seen  in  man  usually 
occurs  from  wounds  into  which  earth  has  been  ground, 
as  in  the  case  of  a  compound,  comminuted  fracture  of  the 
humerus,  with  fatal  infection,  reported  by  Dunham,  or  in 
wounds  and  injuries  in  the  neighborhood  of  the  perineum. 

Among  the  twenty-three  cases  reported  by  Welch  and 
Flexner  *  we  find  wounds  of  the  knee,  leg,  hip,  and  forearm, 
ulcer  of  the  stomach,  typhoid  ulcerations  of  the  intestine, 
strangulated  hernia  with  operation,  gastric  and  duodenal 
ulcer,  perineal  section,  and  aneurysm,  as  conditions  in  which 
external  or  gastro-intestinal  infection  occurred. 

Dobbin,  f  P.  Ernst,  {  Graham,  Stewart  and  Bald  win,  §  and 
Kronig  and  Menge||  have  studied  cases  of  puerperal  sepsis 
and  sepsis  following  abortion  either  caused  by  the  bacillus, 
or  in  which  it  played  an  important  role. 

Williams  **  has  found  the  bacillus  in  a  case  of  suppura- 
tive  pyelitis. 

The  symptoms  following  infection  are  quite  uniform,  con- 
sisting of  redness  and  swelling  of  the  wound,  with  rapid 
elevation  of  temperature  and  rapid  pulse.  The  wound  usu- 
ally becomes  more  or  less  emphysematous,  and  discharges  a 
thin,  dirty,  brownish,  offensive  fluid  that  contains  gas  bubbles 
and  is  sometimes  frothy.  The  patients  occasionally  recover, 
especially  when  the  infected  part  can  be  amputated,  but 
death  is  the  common  outcome.  After  death  the  body  begins 
to  swell  almost  immediately  and  may  attain  twice  its  normal 
size  and  be  unrecognizable.  Upon  palpation  a  peculiar  crep- 
itation can  be  felt  in  the  subcutaneous  tissue  nearly  every- 
where, and  the  presence  of  gas  in  the  blood-vessels  is  easy  of 
demonstration.  The  gas  is  inflammable,  and  as  the  bubbles 
ignite  explosive  sounds  are  heard. 

At  the  autopsy  the  gas  bubbles  are  found  in  most  of  the 
internal  organs,  sometimes  so  numerously  as  to  justify  the 

*  "Journal  of  Experimental  Medicine,"  vol.  I,  No.  1,  Jan.,  1896. 

t"Bull.  Johns  Hopkins  Hospital/'  Feb.,  1897,  No.  71,  p.  24. 

J  "Virchow's  Archiv,"  Bd.  cxxxm,  Heft  2. 

§  "Columbus  Med.  Jour.,"  Aug.,  1893. 

||  "  Bakteriologie  des  weiblichen  Genitalkanals,"  Leipzig,  1897. 

**  "Bull.  Johns  Hopkins  Hospital,"  April,  1896,  p.  66. 


384  Gaseous  Edema 

German  term  " Schaumorgane  "  (frothy  organs).  The  liver 
is  especially  apt  to  show  this  condition.  When  such  tissues 
are  hardened  and  examined  microscopically,  the  bubbles 
appear  as  spaces  in  the  tissue,  their  borders  lined  with  large 
numbers  of  the  bacillus.  There  are  also  clumps  of  bacilli 
without  gas  bubbles,  but  surrounded  by  tissue,  whose 
nuclei  show  a  disposition  to  fragment  or  disappear,  and 
whose  cells  and  fibers  show  signs  of  disintegration  and 
fatty  change.  In  discussing  these  changes  Ernst  concluded 
that  they  were  ante-mortem  and  due  to  the  irritation  caused 
by  the  bacillus.  The  gas-production  he  regards  as  post- 
mortem. 

In  the  internal  organs  the  bacillus  is  usually  found  in  pure 
culture,  but  in  the  wound  it  is  usually  mixed  with  other  bac- 
teria. On  this  account  it  is  difficult  to  estimate  just  how 
much  of  the  damage  before  death  depends  upon  the  activity 
of  the  gas  bacillus.  That  gas-production  after  death  has 
nothing  to  do  with  pathogenesis  during  life  is  shown  by 
injecting  into  the  ear-vein  of  a  rabbit  a  liquid  culture  of  the 
gas  bacillus,  permitting  about  five  minutes'  time  for  the  dis- 
tribution of  the  bacilli  throughout  the  circulation,  and  then 
killing  the  rabbit.  In  a  few  hours  the  rabbit  will  swell  and 
its  organs  and  tissues  be  riddled  with  the  gas  bubbles. 

At  times,  however,  as  in  a  case  of  Graham,  Stewart  and 
Baldwin,  there  is  no  doubt  but  that  the  bacillus  produces 
gas  in  the  tissues  of  the  body  during  life.  These  observers, 
in  a  case  of  abortion  with  subsequent  infection,  found  the 
patient  "emphysematous  from  the  top  of  her  head  to  the 
soles  of  her  feet"  several  hours  before  death. 

In  this  case,  in  which  the  bacillus  was  found  in  pure  culture, 
it  would  indeed  be  difficult  to  doubt  that  the  fatal  issue  was 
due  to  Bacillus  aerogenes  capsulatus. 

Probably  the  best  review  of  the  subject  is  to  be  found  in 
"A  Contribution  to  the  Knowledge  of  the  Bacillus  Aerog- 
enes Capsulatus,"  by  W.  T.  Howard,  Jr.* 

*  "Contributions  to  the  Science  of  Medicine  by  the  Pupils  of  W.  H. 
Welch,"  1900,  p.  461. 


CHAPTER   III. 
TETANUS* 

BACILLUS  TETANI  (FLUGGE). 

General  Characteristics. — A  motile,  flagellated,  sporogenous, 
liquefying,  obligatory  anaerobic,  non-chromogenic,  toxic,  pathogenic 
bacillus  of  the  soil,  staining  by  ordinary  methods  and  by  Gram's 
method.  Its  chief  morphologic  characteristic  is  the  occurrence  of  a 
large  round  spore  at  one  end. 

The  bacillus  of  tetanus  was  discovered  by  Nicolaier  *  in 
1884,  and  obtained  in  pure  culture  by  Kitasatof  in  1889. 
It  is  universally  acknowledged  to  be  the  cause  of  tet- 
anus. 

Distribution. — The  tetanus  bacillus  is  a  common  sapro- 
phyte in  garden  earth,  dust,  and  manure,  and  is  a  constant 
parasite  in  the  intestinal  canal  of  herbivorous  animals. 

The  relation  of  the  bacillus  to  manure  is  interesting,  but 
it  is  most  probable  that  manured  ground,  because  it  is 
richer,  permits  the  bacilli  to  flourish  better  than  sterile 
ground.  The  common  occurrence  of  the  bacilli  in  the 
excrement  of  herbivorous  animals  is  to  be  explained  through 
the  accidental  ingestion  of  earth  with  the  food  cropped  from 
the  ground.  The  spores  of  the  bacillus  thus  reaching  the 
intestine,  seem  able  to  develop  because  of  appropriate 
anaerobic  conditions. 

Verneuil  has  observed  that  tetanus  rarely  occurs  at  sea 
except  upon  cattle  transports,  where  there  is  likely  to  be 
considerable  earth  and  dust  from  hay,  straw,  etc.,  which 
may  carry  the  bacilli. 

Le  Dantec  t  has  shown  that  the  tetanus  bacillus  is  a 
common  organism  in  New  Hebrides,  where  the  natives  poison 
their  arrows  by  dipping  them  into  a  clay  rich  in  tetanus 
bacilli. 

*"  Deutsche  med.  Wochenschrift,"  1884,  42. 

t/Wd.,  1889,  No.  31. 

J  See  abstracts  in  the  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  IX. 
286;  xm,  351. 

25  385 


386  Tetanus 

Morphology. — The  tetanus  bacillus  is  a  long,  slender 
organism  measuring  0.3  to  0.5  X  2  to  4^  (Fliigge).  Its  most 
striking  characteristic  is  an  enlargement  of  one  end,  which 
contains  a  large  round  spore.  The  bacilli  in  which  no 
spores  are  yet  formed  have  rounded  ends  and  seldom  unite 
in  chains  or  pairs.  They  are  motile  and  have  many  flagella 
arising  from  all  parts  of  the  surface  (petrichia). 

Staining. — The  bacilli  stain  readily  with  ordinary  aqueous 
solutions  of  the  anilin  dyes  and  by  Gram's  method. 


Fig.  117.— Bacillus  tetani.     X  1000  (Frankel  and  Pfeiffer). 

Isolation. — The  method  usually  employed  for  the  isolation 
of  the  tetanus  bacillus  was  originated  by  Kitasato,  and  based 
upon  the  observation  that  its  spores  can  resist  exposure  to 
high  temperatures  for  considerable  periods  of  time.  After 
finding  by  microscopic  examination  that  the  bacilli  were 
present  in  pus,  Kitasato  spread  it  upon  the  surface  of  an 
ordinary  agar-agar  tube  and  incubated  it  for  twenty-four 
hours,  during  which  time  all  of  the  contained  micro-organ- 
isms, including  the  tetanus  bacillus,  increased  in  number. 
He  then  exposed  it  for  an  hour  to  a  temperature  of  80°  C., 
by  which  all  fully  developed  bacteria,  tetanus  as  well  as  the 
others,  and  the  great  majority  of  the  spores,  were  destroyed. 
As  scarcely  anything  but  the  tetanus  spores  remained  alive, 
their  subsequent  growth  gave  a  fairly  pure  culture. 

Cultivation. — The  tetanus  bacillus  is  difficult  to  culti- 


Cultivation 


387 


vate  because  it  will  not  grow  where  the  smallest  amount 
of  free  oxygen  is  present.  It  is  hence  a  typical  obligatory 
anaerobe.  Farran  *  and  Grixoni  believe  it  to  have  originally 
been  an  optional  anaerobe,  and  it  is  said  by  these  writers 
that  the  organism  can  gradually  be  accustomed  to  oxygen 
so  as  to  grow  in  its  presence.  When  this  is  achieved,  it 
loses  its  virulence. 


Fig.  1 1 8.— Bacillus  tetani;  six-  Fig.  119. — Bacillus  tetani;  cul- 

days-old  puncture  culture  in  glu-     ture    four   days    old    in    glucose- 
cose-gelatin(FrankelandPfeiffer).     gelatin  (Frankel  and  Pfeiffer). 

The  methods  for  excluding  the  oxygen  from  the  cul- 
tures and  replacing  it  by  hydrogen,  as  well  as  other  methods 
suggested  for  the  cultivation  of  the  strictly  anaerobic 
organisms,  are  given  under  the  appropriate  heading  (Anae- 
robic Cultures),  and  need  not  be  repeated  here. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  July  15,  1898,  p.  28. 


388  Tetanus 

Park,*  following  the  suggestion  of  Kitasato,  covers  the 
surface  of  the  bouillon  with  a  layer  of  paraffin  about  i  to  2  cm. 
thick.  This  melts  in  the  sterilization  and  forms  a  firm  layer, 
through  which  the  bouillon  is  inoculated,  warmed  until 
the  paraffin  melts  again,  then  stood  away  until  develop- 
ment in  the  air-free  bouillon  occurs.  If  the  paraffin  be 
found  too  brittle,  some  albalene  may  be  mixed  with  it  until 
it  is  flexible  when  cool. 

The  colonies  of  the  tetanus  bacillus,  when  grown  upon 
gelatin  plates  in  an  atmosphere  of  hydrogen,  resemble  those 


Fig.  120. — Bacillus  tetani;  five-day-old  colony  upon  gelatin-containing 
glucose.      X  1000  (Frankel  and  Pfeiffer). 

of  the  well-known  hay  bacillus.  There  is  a  rather  dense, 
opaque  central  mass  surrounded  by  a  more  transparent 
zone,  the  margins  of  which  consist  of  a  fringe  of  radially 
projecting  bacilli.  Liquefaction  occurs  slowly. 

Bouillon. — The  organism  can  be  grown  in  bouillon,  and 
attains  its  maximum  development  at  a  temperature  of 
37°  C.  Gas  is  given  off  from  the  cultures,  and  they  have  a 
peculiar  odor,  very  characteristic,  but  difficult  to  describe, 
The  bouillon  is  clouded  and  contains  a  sediment. 

*  "Jour.  Med.  Research,"  N.  S.,  vol.  i,  No.  i,  p.  298. 


Vital  Resistance 


389 


In  bouillon  containing  sugar  considerable  gas  is  formed  in 
the  fermentation  tube.    Both  CO2  and 
H2S  are  formed 

Gelatin. — The  growth  occurs  deep 
in  the  puncture,  and  is  arborescent. 
Liquefaction  begins  in  the  second 
week  and  causes  the  disappearance  of 
the  radiating  filaments.  The  lique- 
faction spreads  slowly,  but  may  in- 
volve the  entire  mass  of  gelatin  and 
resolve  it  into  a  grayish-white  syrupy 
liquid,  at  the  bottom  of  which  the 
bacilli  accumulate.  The  growth  in 
gelatin  containing  glucose  is  rapid. 

Agar-agar. — The  growth  in  agar- 
agar  punctures  is  slower,  but  similar 
to  the  gelatin  cultures  except  for  the 
absence  of  liquefaction. 

Milk  is  favorable  for  the  develop- 
ment of  the  tetanus  bacillus.  There 
is  no  coagulation.  Litmus  milk  is 
acidified. 

Potato. — Upon  potatoes  under  strict 
anaerobic  conditions  the  bacilli  grow 
but  slightly. 

Vital  Resistance. — The  tetanus 
spores  may  remain  alive  in  dry  earth 
for  many  years.  Sternberg  says  they 
can  resist  immersion  in  5  per  cent, 
aqueous  carbolic  acid  solutions  for  ten 
hours,  but  fail  to  grow  after  fifteen 
hours.  A  5  per  cent,  carbolic  acid 
solution,  to  which  0.5  per  cent,  of 
hydrochloric  acid  has  been  added, 
destroys  them  in  two  hours.  They 
are  destroyed  in  three  hours  by 
i  :  looo  bichlorid  of  mercury  solu- 
tion, but  when  to  such  a  solution 
0.5  per  cent,  of  hydrochloric  acid  is 
added,  its  activity  is  so  increased  that 
the  spores  are  destroyed  in  thirty 
minutes.  According  to  Kitasato,*  ex- 
posure to  streaming  steam  for  from  five  to  eight  minutes  is 
*  "Zeitschrift  fur  Hygiene,"  xn,  p.  225. 


Fig.  121 . — Tetanus 
bacillus ;  glucose-agar 
culture,  five  months  old 
(Curtis). 


390  Tetanus 

certain  to  kill  tetanus  spores,  and  this  statement  has  found 
its  way  into  most  of  the  text-books  without  discussion. 
Theobald  Smith,*  however,  has  studied  several  cultures 
of  the  organism  and  finds  that  its  resistance  to  heat  is  much 
greater,  and  that  in  one  case  seventy  minutes'  exposure  to 
streaming  steam  did  not  kill  all  of  the  spores. 

Metabolic  Products. — Bouillon  cultures  of  the  tetanus 
bacillus  contain  acids,  proteolytic  ferment,  and  several  toxic 
substances,  of  which  tetanospasmin  and  tetanolysin  are  best 
known.  The  toxic  products  are  apparently  all  soluble. 
No  endotoxin  is  known  to  be  formed. 

The  most  ready  method  of  preparing  the  toxins  for  experi- 
mental study  is  to  cultivate  the  bacilli  in  freshly  prepared 
neutral  or  slightly  alkaline  sugar-free  bouillon  under  condi- 
tions of  most  strict  anaerobiosis,  at  a  temperature  of  37° 
C.,  and  then  filter  the  culture  through  porcelain.  Field  f 
found  the  highest  degree  of  toxicity  about  the  sixth  or 
seventh  day.  It  may  attain  a  toxicity  so  great  that 
0.000005  c-c-  will  cause  the  death  of  a  mouse.  I  found  the 
average  toxicity  such  that  o.ooi  c.c.  was  fatal  to  a  guinea- 
pig.  KnorrJ  gives  some  interesting  comparisons  of  the 
susceptibility  of  different  animals,  as  follows: 

i  gram  of  horse  is  destroyed  by x  toxin 

i  gram  of  goat  is  destroyed  by 2x  toxin 

i  gram  of  mouse  is  destroyed  by 13  x  toxin 

i  gram  of  rabbit  is  destroyed  by 2,000  x  toxin 

i  gram  of  hen  is  destroyed  by 200,000  x  toxin 

The  toxin  is  very  unstable,  and  is  easily  destroyed  by 
heat  above  60°  C.  It  is  also  quickly  destroyed  by  light, 
especially  direct  sunlight.  Flexnerand  Noguchi§  found  that 
5  per  cent,  of  eosin  added  to  the  toxin  destroyed  it  through 
the  photodynamic  power  of  the  stain.  The  toxin  is  also 
easily  destroyed  by  electric  currents.  It  is  also  decomposed 
by  exposure  to  the  air  and  light,  so  that  it  is  difficult  to  pre- 
serve it  for  many  days.  The  best  method  of  keeping  it  is  to 
add  0.5  per  cent,  of  phenol,  and  then  store  it  in  a  cool,  dark 
place,  in  bottles  completely  filled  and  tightly  corked.  It  will 
not  keep  its  strength  in  liquid  form  under  the  best  conditions. 

*  "Jour.  Amer.  Med.  Assoc.,"  March  21,  1908,  vol.  L,  No.  12,  p.  931. 
t  "Proc.  N.  Y.  Path.  Soc.,"  March,  1904,  p.  18. 
J  "Munch,  med.  Wochenschrift,"  1898,  p.  321. 
§  "Studies  from  the  Rockefeller  Institute,"  1905,  v. 


Metabolic  Products  391 

To  keep  it  for  experimental  purposes  it  is  advisable 
to  precipitate  it  by  supersaturation  with  ammonium  sul- 
phate, which  causes  it  to  float  upon  the  liquid  in  the  form 
of  a  sticky  brown  scum.  It  can  be  skimmed  oft7  and  dried. 
Such  dry  precipitate  will  retain  its  activity  for  months 
with  but  little  deterioration. 

From  cultures  of  tetanus  bacilli  grown  in  various  media, 
and  from  the  blood  and  tissues  of  animals  affected  with  the 
disease,  Brieger  succeeded  in  separating  "  tetanin,"  "  tetano- 
toxin,"  "  tetanospasmin,"  and  a  fourth  substance  to  which 
no  name  is  given.  All  were  very  poisonous  and  productive 
of  tonic  convulsions.  Later  Brieger  and  Frankel  isolated  an 
extremely  poisonous  toxalbumin  from  sugar-bouillon  cul- 
tures of  the  bacillus.  Ehrlich*  later  discovered  a  new  poison- 
ous element  to  which  he  applied  the  name  tetanolysin. 

The  purified  toxin  of  Brieger  and  Cohn  was  surely  fatal 
to  mice  in  doses  of  0.00000005  gram.  The  work  of  these 
older  writers  is  now  so  completely  superseded  by  that  of 
others  as  to  be  of  historic  interest  only.  Lambert  f  considers 
the  tetanus  toxin  to  be  the  most  poisonous  substance  that 
has  ever  been  discovered. 

Fermi  and  PernossJ  found  most  toxin  produced  in  agar- 
agar  cultures,  less  in  gelatin  cultures,  and  least  in  bouillon 
cultures. 

Ehrlich  §  found  two  poisons  in  the  tetanus  toxin,  one  of 
which  was  convulsive  and  was  in  consequence  called  tetano- 
spasmin, the  other  hemolytic  and  called  tetanolysin.  When 
tetanus  toxin  is  added  to  defibrinated  blood,  the  tetano- 
lysin is  absorbed  by  the  corpuscles,  many  of  which  are 
dissolved,  while  the  tetanospasmin  remains  unchanged. 

D6nitz||  and  Wassermann  and  Takaki**  have  found  that 
the  tetanus  toxin  has  a  specific  affinity  for  the  central 
nervous  system,  with  whose  cells  it  combines  in  vitro  and 
becomes  inert. 

Roux  and  Borreljt  have  also  found  that  when  tetanus 
toxin  is  injected  into  the  brain  substance  a  very  much 

*  "jBerliner  klin.  Wochenschrift,"  1898. 

f  "New  York  Med.  Jour.,"  June  5,  1897. 

t  "Centralbl.  f.  Bakt.,"  etc.,  xv,  p.  303. 

§  "Berliner  klin.  Wochenschrift,"  1898,  No.  12,  p.  273. 

§  "Deutsche  med.  Wochenschrift,"  1897,  p.  428. 
**  "Berliner  klin.  Wochenschrift,"  1898,  35. 
ft  "Ann.  de  1'Inst.  Pasteur,"  t.  xn,  1898. 


39  2  Tetanus 

smaller  dose  will  cause  death  than  is  necessary  when  the 
poison  is  absorbed  from  the  subcutaneous  tissues. 

Like  most  of  the  bacterial  toxins,  the  tetanus  poison  is 
only  effective  when  produced  in  or  injected  into  the  tissues 
and  absorbed  into  the  circulation.  It  is  harmless  when 
given  by  the  digestive  tract,  Ramon*  having  adminis- 
tered by  the  mouth  300,000  times  the  fatal  hypodermic 
dose  without  producing  any  symptoms.  The  toxin  seemed 
to  pass  out  with  the  feces. 

One  of  the  most  interesting  peculiarities  about  the  toxin 
is  the  comparative  uniformity  of  the  period  intervening 
between  its  administration  and  the  appearance  of  the 
symptoms — erroneously  called  the  incubation  period.  This 
varies  within  a  narrow  margin,  inversely,  with  the  size  of 
the  dose.  Thus,  according  to  Behring,  the  effect  of  varying 
doses  of  the  toxin  upon  mice  becomes  evident  according 
to  the  size  of  the  dose  in  from  twelve  to  thirty-six  hours, 
thus: 

13  lethal  doses symptoms  in  36  hours 

1 10  lethal  doses symptoms  in  24  hours 

333  lethal  doses symptoms  in  20  hours 

1300  lethal  doses symptoms  in  14  hours 

3600  lethal  doses symptoms  in  12  hours 

The  local  action  of  the  toxin  is  very  painful  and  asso- 
ciated with  spasm  of  the  muscular  fibers  with  which  it 
comes  in  contact.  Pitfield,f  thinking  that  it  might  be 
useful  in  the  treatment  of  certain  paralytic  affections, 
injected  a  minute  quantity  of  it  into  the  calf  of  his  leg 
and  experienced  the  severe  spasmodic  local  effects  of  the 
poison  for  twelve  hours. 

It  has  been  the  belief  of  most  physiologists  that  tetanus 
toxin  acts  solely  upon  the  motor  cells  of  the  spinal  cord,  and 
produced  the  tonic  spasms  as  strychnin  does.  The  affinity 
of  the  toxin  for  the  nervous  tissues  has  been  made  the 
subject  of  careful  investigations  by  Marie  and  Moraxf  and 
Meyer  and  Ransom.  §  The  former  found  that  the  absorption 
of  tetanus  toxin  took  place  partly  through  the  peripheral 
nerves  because  of  specific  affinity  between  the  toxin  and 

*  "  Deutsche  med.  Wochenschrift,"  Feb.  24,  1898. 
t  "Therapeutic  Gazette,"  March  15,  1897. 

J  "Ann.  de  1'Inst.  Pasteur,"  1902,  xvi,  p.  818;  and  "Bull,  de  1'Inst. 
Past.,"  1903,  i,  p.  41. 

§  "Arch.  f.  exper.  Path.  u.  Pharmak.,"  1903,  xux. 


Metabolic  Products  393 

the  axis  cylinder  substance;  the  latter  found  the  toxin  car- 
ried to  the  central  nervous  system  solely  by  the  motor 
nerves,  the  action  depending  upon  the  integrity  of  the 
axis  cylinder.  They  believe  that  the  toxin  is  absorbed 
by  the  axis  cylinder  endings,  and  reaching  the  correspond- 
ing spinal  nerve  center  by  that  route  spreads  to  the  cor- 
responding center  in  the  other  half  of  the  cord  and  out- 
ward, resulting  in  generalized  tetanus.  When  intoxication 
is  produced  through  the  circulation,  the  poison  is  taken 
up  by  the  nerve  endings  in  all  parts  of  the  body,  and  the 
disease  is  not  localized,  but  general.  Antitoxin,  unlike  the 
toxin,  does  not  travel  by  the  nerve  route,  but  is  found  only 
in  the  blood  and  lymph.  Zupnik*  has  brought  forward 
evidence  that  this  view  is  incorrect  and  that  there  are 
two  distinct  actions  caused  by  the  toxin.  He  differentiates 
between  tetanus  ascendens  and  tetanus  descendens.  The 
former  always  succeeds  the  intramuscular  introduction  of 
the  toxin,  and  depends  upon  its  direct  action  upon  the 
muscle  itself.  It  explains  the  familiar  phenomenon  of 
rigidity  making  its  first  appearance  in  that  member  into 
which  the  inoculation  was  made.  The  ascending  tetanus 
gradually  ascends  from  muscle  to  muscle.  He  thinks  the 
absorption  of  the  poison  by  the  muscle-cells  depends  upon 
their  normal  metabolic  function,  as  when  their  nerves  are 
severed,  the  fixation  of  the  toxin  and  the  occurrence  of  the 
tonic  spasm  does  not  occur. 

Tetanus  descendens  results  from  the  entrance  of  the  toxin 
into  the  circulation  from  the  cellular  tissue  and  its  distribu- 
tion in  the  blood.  Under  these  conditions  Zupnik  believes 
it  acts  upon  the  central  nervous  system,  especially  upon  the 
spinal  cord,  manifesting  itself  in  extreme  reflex  excitability 
with  irregular  motor  discharges  resulting  in  clonic  spasms. 

There  are,  therefore,  two  forms  of  spasm  in  tetanus: 
the  tonic  convulsions,  seeming  to  depend  upon  local  action 
and  fixation  of  the  toxin,  and  the  clonic  convulsions,  de- 
pending upon  the  centric  action.  The  latter  are  the  more 
dangerous  for  the  sufferer. 

The  lockjaw  or  trismus  and  the  opisthotonos  that  are  so 
characteristic  of  the  affection  depend,  according  to  Zupnik's 
view,  upon  a  loss  of  equilibrium  among  the  muscles  affected. 
They  occur  only  in  descending  tetanus  and  depend  upon 
spasm  of  muscle  without  equally  powerful  opposing  groups. 
*  "Wiener  klin.  Wochenschrift,"  Jan.  23,  1902. 


394  Tetanus 

The  stronger  muscles  of  the  jaw  are  those  that  close  it;  the 
stronger  muscles  of  the  back,  those  of  the  erector  group. 
This  view  is  exactly  the  opposite  of  Meyer  and  Ransom,* 
who  believe  that  the  tetanus  toxin  is  absorbed  only  along 
the  nerve  trunks,  and  found  that  section  of  the  spinal  cord 
prevented  the  ascent  of  tetanus  from  the  lower  extremities. 
Injection  of  the  toxin  into  a  posterior  nerve-root  produced 
tetanus  dolorosus.  Injection  of  the  toxin  into  a  posterior 
nerve-root  together  with  section  of  the  spinal  cord  produced 
exaltation  of  the  reflex  irritability — "Jactationstetanus." 
Injection  in  sensory  nerves  does  not  produce  tetanus  doloro- 
sus because  the  transportation  of  the  poison  along  these 
trunks  is  so  slow. 

The  tetanolysin  is  a  hemolytic  component  of  the  toxic 
bouillon,  and  is  entirely  separate  and  distinct  from  the  tetano- 
spasmin  or  convulsive  poison.  It  probably  takes  no  part  in 
the  usual  clinical  manifestations  of  tetanus. 

Pathogenesis. — The  work  of  Kitasato  has  given  us  very 
complete  knowledge  of  the  biology  of  the  tetanus  bacillus 
and  completely  established  its  specific  nature: 

When  a  white  mouse  is  inoculated  with  an  almost  in- 
finitesimal amount  of  tetanus  culture,  or  with  garden  earth 
containing  the  tetanus  bacillus,  the  first  symptoms  come 
on  in  from  one  to  two  days,  when  the  mouse  develops 
typical  tetanic  convulsions,  first  beginning  in  the  neighbor- 
hood of  the  inoculation,  but  soon  becoming  general.  Death 
follows  sometimes  in  a  very  few  hours.  In  rabbits,  guinea- 
pigs,  mice,  rats,  and  other  small  animals  the  period  of 
incubation  is  from  one  to  three  days.  In  man  the  period 
of  incubation  varies  from  a  few  days  to  several  weeks,  and 
averages  about  nine  days. 

The  disease  is  of  much  interest  because  of  its  purely 
toxic  nature.  There  is  usually  a  small  wound  with  a  slight 
amount  of  suppuration  and  at  the  autopsy  the  organs  of  the 
body  are  normal  in  appearance,  except  the  nervous  system, 
which  bears  the  greatest  insult.  It,  however,  shows  little  else 
than  congestion  either  macroscopically  or  microscopically. 

The  conditions  in  the  animal  body  are  in  general  un- 
favorable to  the  development  of  the  bacilli,  because  of  the 
loosely  combined  oxygen  contained  in  the  blood,  and  they 
grow  with  great  slowness,  remaining  localized  at  the  seat 
of  inoculation,  and  never  entering  the  blood.  Doubtless 

t  "  Archiv.  f.  exper.  Path.  u.  Pharmak.,"  Bd.  xux,  1903,  p.  396. 


Pathogenesis  395 

most  cases  of  tetanus  are  cases  of  mixed  infection  in  which 
the  bacillus  enters  with  aerobic  bacteria,  which  aid  its 
growth  by  absorbing  the  oxygen  in  the  neighborhood.  The 
amount  of  poison  produced  must  be  exceedingly  small  and 
its  power  tremendous,  else  so  few  bacilli  growing  under 
adverse  conditions  could  not  produce  fatal  toxemia.  The 
toxin  is  produced  rapidly,  for  Kitasato  found  that  if  mice 
were  inoculated  at  the  root  of  the  tail,  and  the  skin  and 
the  subcutaneous  tissues  around  the  inoculation  afterward 
either  excised  or  burned  out,  the  treatment  would  not  save 
the  animal  unless  the  operation  were  performed  within 
an  hour  after  the  inoculation. 

Some  incline  to  the  view  that  the  toxin  is  a  ferment, 
and  the  experiments  of  Nocard*  might  be  adduced  in 
support  of  the  theory.  He  says:  "Take  three  sheep  with 
normal  tails,  and  insert  under  the  skin  at  the  end  of  each 
tail  a  splinter  of  wood  covered  with  the  dried  spores  of  the 
tetanus  bacillus";  watch  these  animals  carefully  for  the  first 
symptoms  of  tetanus,  then  amputate  the  tails  of  two  of 
them  20  cm.  above  the  point  of  inoculation,  .  .  .  the 
three  animals  succumb  to  the  disease  without  showing  any 
sensible  difference." 

The  circulating  blood  of  diseased  animals  is  fatal  to 
susceptible  animals  because  of  the  toxin  which  it  contains; 
and  the  fact  that  the  urine  is  also  toxic  to  mice  proves  that 
the  toxin  is  excreted  by  the  kidneys. 

The  organisms  usually  enter  the  body  through  a  wound 
caused  by  some  implement  which  has  been  in  contact  with 
the  soil,  or  enter  abrasions  from  the  soil  directly.  Doubt- 
less many  of  the  wounds  are  so  small  that  their  existence  is 
overlooked,  and  this,  together  with  the  fact  that  the  period 
of  incubation  of  the  disease,  especially  in  man,  is  of  con- 
siderable duration  (three  to  nine  days),  and  at  times  permits 
the  wound  to  heal  before  any  symptoms  of  intoxication 
occur,  serves  to  explain  the  occurrence  of  some  of  the 
reported  cases  in  which  no  wound  is  said  to  have  existed. 

There  are  two  classes  of  infected  wounds  particularly  prone 
to  be  followed  by  tetanus — namely,  those  into  which  soil 
has  been  carried  by  the  injuring  implement  and  those  of 
considerable  depth.  The  infecting  organism  reaches  the 
first  class  in  large  numbers,  but  finds  itself  under  aerobic 
and  other  inappropriate  conditions  of  growth.  It  reaches 
*  Quoted  before  the  Academic  de  Medicine,  Oct.  22,  1895. 


396  Tetanus 

the  second  class  in  smaller  numbers,  but  finds  the  conditions 
of  growth  better  because  of  the  depth  of  the  wound. 

The  severity  of  the  wound  has  nothing  whatever  to  do 
with  the  occurrence  of  tetanus,  pin -pricks,  nail  punctures, 
insect  stings,  vaccination,  and  a  variety  of  other  mild 
injuries  sometimes  being  followed  by  it. 

An  interesting  fact  has  been  presented  by  Vaillard  and 
Rouget,*  who  found  that  if  the  tetanus  spores  were  intro- 
duced into  the  body  freed  from  their  poison,  they  were 
unable  to  produce  the  disease  because  of  the  promptness 
with  which  the  phagocytes  took  them  up.  If,  however, 
the  toxin  was  not  removed,  or  if  the  body-cells  were  injured 
by  the  simultaneous  introduction  of  lactic  acid  or  other 
chemic  agent,  the  spores  would  immediately  develop  into 
bacilli,  begin  to  manufacture  toxin,  and  produce  the  disease. 
This  suggests  that  many  wounds  may  be  infected  by  the 
tetanus  bacillus  though  the  surrounding  conditions  rarely 
enable  it  to  develop  satisfactorily  and  produce  enough 
toxin  to  cause  disease. 

In  very  rare  cases  tetanus  may  possibly  occur  without 
the  previous  existence  of  a  wound,  as  in  the  case  reported 
by  Kamen,  who  found  the  intestine  of  a  person  dead  of 
the  disease  rich  in  Bacillus  tetani.  Kamen  is  of  the  opinion 
that  the  bacilli  can  grow  in  the  intestine  and  be  absorbed, 
especially  where  imperfections  in  the  mucosa  exist.  It  is 
not  impossible,  though  he  does  not  think  it  probable,  that 
the  bacteria  growing  in  the  intestine  can  elaborate  enough 
toxin  to  produce  the  disease  by  absorption. 

A  peculiar  observation  has  been  made  by  Montesano  and 
Montesson,  *  who  unexpectedly  found  the  tetanus  bacillus 
in  pure  culture  in  the  cerebro-spinal  fluid  of  a  case  of  par- 
alytic dementia  that  died  without  a  tetanic  symptom. 

Immunity. — All  animals  are  not  alike  susceptible  to 
tetanus.  Men,  horses,  mice,  rabbits,  and  guinea-pigs  are 
susceptible;  dogs  much  less  so.  Cattle  suffer  chiefly  after 
accouchement,  and  after  abortion.  Most  birds  are  scarcely  at 
all  susceptible  either  to  the  bacilli  or  to  their  toxin.  Am- 
phibians and  reptiles  are  immune,  though  it  is  said  that  frogs 
can  be  made  susceptible  by  elevation  of  their  body-tempera- 
ture. 

*  See  "  Centralbl.  f.  Bakt.,  Infekt.,  u.  Parasitenk.,"  vol.  xvi,  p.  208. 
t"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Dec.,  1897,  Bd.  xxn,  Nos. 
22,  23,  p.  663. 


Antitoxin  397 

The  injection  of  the  toxic  bouillon  or  of  the  redis- 
solved  ammonium  sulphate  precipitate,  in  progressively 
increasing  doses,  into  animals,  causes  the  formation  of  anti- 
bodies (antitoxin)  by  which  the  effects  of  both  the  tetano- 
spasmin  and  the  tetanolysin  are  destroyed.  The  purely  toxic 
character  of  the  disease  makes  it  peculiarly  well  adapted  for 
treatment  with  antitoxin,  and  at  the  present  time  our  sole 
therapeutic  reliance  is  placed  upon  it.  The  mode  of  prepar- 
ing the  serum  and  the  system  of  standardization  are  discussed 
in  the  section  upon  Antitoxins  in  the  part  of  this  work  that 
treats  of  the  Special  Phenomena  of  Infection  and  Immunity. 

Antitoxin. — Numerous  cases  of  the  beneficial  action  of 
antitoxin  are  on  record,  but,  as  Welch*  has  pointed  out, 
the  antitoxin  of  tetanus  has  proved  a  disappointment  in 
the  treatment  of  tetanus.  Moschcowitz,f  in  his  excellent 
literary  review  of  the  subject,  has  shown  that  its  use  has 
reduced  the  death-rate  from  about  80  to  40  per  cent.,  and 
that  it  therefore  cannot  be  looked  upon  as  a  failure.  The 
result  of  its  experimental  injection,  in  combination  with 
the  toxin,  into  mice,  guinea-pigs,  rabbits,  and  other  animals 
is  perfectly  satisfactory,  and  affords  protection  against 
almost  any  multiple  of  the  fatal  dose,  but  the  quantity 
needed,  in  proportion  to  the  body-weight,  to  save  an  animal 
from  the  unknown  quantity  of  toxin  being  manufactured 
in  its  body  increases  so  enormously  with  the  day  or  hour 
of  the  disease  as  to  make  the  dose,  which  increases  millions 
of  times  where  that  of  diphtheria  antitoxin  increases  but 
tenfold,  a  matter  of  difficulty  and  uncertainty.  Nocard 
also  called  attention  to  the  fact  that  the  existence  of  tetanus 
cannot  be  known  until  a  sufficient  toxemia  to  produce 
spasms  exists,  and  that  therefore  it  is  impossible  to  attack 
the  disease  in  its  inception  or  to  begin  the  treatment  until  too 
late  to  effect  a  cure.  At  this  point  it  is  well  to  recall 
Nocard's  experiment  with  the  sheep,  in  whose  blood  so  much 
toxin  was  already  present  when  symptoms  first  appeared 
that  the  amputation  of  their  infected  tails  could  not  save 
them. 

The  explanation  of  this  inability  of  the  antitoxin  to  effect 
a  cure  when  administered  after  development  of  the  symp- 
toms of  tetanus  is  probably  found  in  a  ready  fixation  of  the 

*  "Bulletin  of  the  Johns  Hopkins  Hospital,"  July  and  August, 
1895. 

f  "Annals  of  Surgery,"  1900,  xxxn,  2,  pp.  219,  416,  567. 


398  Tetanus 

toxin  in  the  bodies  of  the  infected  animals.  This  is  well 
shown  by  the  experiments  of  Donitz,*  who  found  that  if  a 
mixture  of  toxin  and  antitoxin  were  made  before  injection 
into  an  animal,  twelve  minimum  fatal  doses  were  neutralized 
by  i  c.c.  of  a  i  :  2000  dilution  of  an  antitoxin.  If,  however, 
the  antitoxin  was  administered  four  minutes  after  the 
toxin,  i  c.c.  of  a  i  :  600  dilution  was  required;  if  eight 
minutes  after,  i  c.c.  of  a  i  :  200  dilution ;  if  fifteen  minutes 
after,  i  c.c.  of  a  i  :  100  dilution.  He  found  that  similar 
but  slower  fixation  occurred  with  diphtheria  toxin. 

It  was  found  by  Roux  and  Borrel  f  that  doses  of  tetanus 
antitoxin  absolutely  powerless  to  affect  the  progress  of  the 
disease,  when  administered  in  the  ordinary  manner  by 
subcutaneous  injection,  readily  saved  the  animal  if  the 
antitoxin  were  injected  into  the  brain  substance. 

Chauffard  and  Quenu,{  who  injected  the  antitoxin  into 
the  cerebral  substance,  found  that  such  administration 
brought  about  an  apparent  cure  in  one  case. 

Their  observations  were  followed  by  an  attempt  to  apply 
the  method  in  human  medicine,  and  patients  with  tetanus 
were  trephined  and  the  antitoxin  injected  beneath  the  dura 
and  into  the  cerebral  substance.  The  results  have  not,  how- 
ever, been  satisfactory,  and  as  the  method  cannot  be  looked 
upon  as  itself  free  from  danger,  it  has  been  abandoned. 

The  only  means  of  treating  the  disease  that  can  be  recom- 
mended at  present  is  by  intravenous  and  subcutaneous  injec- 
tion of  large  and  frequently  repeated  doses  of  the  antitoxic 
serum.  There  can  be  little  doubt  but  that  the  administra- 
tion must  be  so  free  as  to  load  up  the  patient's  blood  with  the 
antitoxin  in  hopes  that  its  presence  there  may  be  able  to  de- 
tach the  toxic  molecules  from  their  anchorage  to  the  nerve 
cells  and  form  an  inert  union. 

Prophylactic  Treatment. — While  tetanus  antitoxin  is 
extremely  disappointing,  in  practice,  for  the  cure  of  tetanus, 
it  is  most  satisfactory  for  its  prevention.  "  An  ounce  of 
prevention  is  better  than  a  pound  of  cure,"  and  if  the  sur- 
geon would  administer  a  prophylactic  injection  of  tetanus 
antitoxin  in  every  case  in  which  the  occurrence  of  tetanus 
was  at  all  likely,  the  disease  would  rarely  develop. 

*  Reference  18,  in  "Jour,  of  Hygiene,"  vol.  n,  No.  2,  in  Ritchie's 
article. 

t  "Ann.  de  1'Inst.  Pasteur,"  1898,  No.  4. 
t  "  La  Presse  med.,"  No.  5,  1898. 


Bacilli  Resembling  the  Tetanus  Bacillus       399 


BACILLI  RESEMBLING  THE  TETANUS  BACILLUS. 

Tavel  *  has  called  attention  to  a  bacillus  commonly  found 
in  the  intestine,  sometimes  in  large  numbers  in  the  appendix 
in  cases  of  appendicitis,  and  looked  upon  by  one  of  his 
colleagues,  Fraulein  Dr.  von  Mayer,  as  the  probable  common 
cause  of  appendicitis.  He  calls  it  the  "Pseudo-tetanus- 
bacillus." 

The  bacillus  is  slender  and  measures  0.5  by  5-7  /*,  is 
rather  more  slender  than  the  tetanus  bacillus,  and  its  spores 
are  oval,  situated  at  the  end  of  the  rod,  and  cause  a  slight 
bulging  rather  pointed  at  the  end.  The  bacillus  is  provided 
with  not  more  than  a  dozen  flagella, — usually  only  four  to 
eight,— thus  differing  markedly  from  the  tetanus  bacillus, 
which  has  many.  The  flagella  are  easily  stained  by  Loffler's 
method  without  the  addition  of  acid  or  alkali.  The  organ- 
ism does  not  stain  so  well  by  Gram's  method  as  the  true 
tetanus  bacillus.  The  bacillus  is  a  pure  anaerobe. 

The  growth  in  bouillon  is  rather  more  rapid  than  that  of 
the  tetanus  bacillus.  It  will  not  grow  in  gelatin.  The 
growth  in  agar-agar  is  very  luxuriant  and  accompanied 
by  the  evolution  of  gas.  Upon  obliquely  solidified  agar- 
agar  the  colonies  are  round,  circumscribed,  and  often  en- 
compassed by  a  narrow,  clear  zone,  which  is  often  notched. 
The  organism  grows  in  serum  only  in  a  vacuum.  The  spores 
are  killed  at  80°  C. 

The  organism  produced  no  symptoms  in  mice,  guinea- 
pigs,  and  rabbits  even  when  2-5  c.c.  of  a  culture  were 
subcutaneously  introduced. 

Sanfelice  f  and  L/ubinski  {  have  observed  a  bacillus  in 
earth  and  meat-infusions  that  is  morphologically  and  cul- 
turally like  the  tetanus  bacillus,  but  differs  from  it  in  not 
possessing  any  pathogenic  powers. 

Kruse  §  has  also  described  a  bacillus  much  like  the  tetanus 
micro-organism  that  grows  aerobically.  It  is  not  patho- 
genic. 

*  "Centralbl.  f.  Bakt.,"  etc.,  March  31,  1898,  xxm,  No.  13,  p.  538. 

f  "Zeitschrift  fur  Hygiene,"  vol.  xiv. 

}  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  xvi,  19. 

§  Haggis,  "  Die  Mikroorganismen,"  vol.  n,  p.  267. 


CHAPTER   IV. 
ANTHRAX. 

BACIIXUS  ANTHRACIS  (KOCH). 

General  Characteristics. — A  non-motile,  non-flagellated,  spor- 
ogenous,  liquefying,  non-chromogenic,  pathogenic,  aerobic  bacillus 
staining  by  the  ordinary  methods  and  by  Gram's  method. 

The  disease  of  herbivora  known  as  anthrax,  "splenic  fever," 
"Milzbrand,"  and  "charbon,"  of  infrequent  occurrence  in 
this  country  and  England,  is  a  dreaded  and  common  malady 
in  France,  Germany,  Hungary,  Russia,  Persia,  and  the 
East  Indian  countries.  In  Siberia  the  disease  is  so  common 


Fig.   122. — Bacillus  anthracis;  colony  three  days  old  upon  a  gelatin 
plate;  adhesive  preparation.      X  1000  (Frankel  and  Pfeiffer). 

and  malignant  as  to  deserve  its  popular  name,  "Siberian 
pest."  Certain  districts,  as  the  Tyrol  and  Auvergne,  in 
which  it  seems  to  be  endemic,  serve  as  foci  from  which  the 
disease  spreads  in  summer,  afflicting  many  animals,  and 
ceasing  its  depredations  only  with  the  advent  of  winter.  It 
is  not  rare  in  the  United  States,  where  it  seems  to  be  chiefly 
a  disease  of  the  summer  season. 

400 


Bacillus  Anthracis  401 

The  animals  most  frequently  affected  are  cows  and  sheep. 
Among  laboratory  animals,  white  mice,  house-mice,  guinea- 
pigs,  and  rabbits  are  highly  susceptible;  dogs,  cats,  most 
birds,  and  amphibians  are  immune.  White  rats  are  infected 
with  difficulty.  Man  is  slightly  susceptible,  the  disease  in 
the  human  species  usually  being  a  local  affection — "malig- 
nant carbuncle" — commonly  succeeded  by  a  general  fatal 
infection. 

Anthrax  was  one  of  the  first  infectious  diseases  proved  to 
depend  upon  a  specific  micro-organism.  As  early  as  1849 


Fig.  123. — Bacillus  anthracis;  showing  the  capsules.     From  a  case  of 
human  infection.     Magnified  1000  diameters  (Schwalve). 

Pollender*  discovered  small  rod-shaped  bodies  in  the  blood 
of  animals  suffering  from  anthrax,  but  the  exact  relation 
which  they  bore  to  the  disease  was  not  pointed  out  until 
1863,  when  Davaine,|  by  a  series  of  interesting  experiments, 
proved  their  etiologic  significance  to  most  unbiased  minds. 
The  final  confirmation  of  Davaine's  conclusions  and  actual 
proof  of  the  matter  rested  with  Koch,J  who,  observing  that 
the  bacilli  bore  spores,  cultivated  them  successfully  outside 
the  body,  and  produced  the  disease  by  the  inoculation  of 
pure  cultures. 

*  "Vierteljahrsschr.  fur  ger.  Med.,"  Bd.  vin,  1855. 
t  "Compte-rendu,"  Ivii,  1863. 
t  "Beitrage  zur  Biol.  d.  Blauzen,"  1876.  n. 
26 


402  Anthrax 

Morphology. — The  anthrax  bacillus  is  a  large 'rod-shaped 
organism,  of  rectangular  form,  with  slightly  rounded  cor- 
ners. It  measures  5  to  20  ^  in  length  and  from  i  to 
1.25  ^  in  breadth.  It  has  a  pronounced  tendency  to 
form  long  threads,  in  which,  however,  the  individuals  can 
usually  be  made  out,  the  lines  of  junction  of  the  com- 
ponent bacilli  giving  the  thread  somewhat  the  appear- 
ance of  a  bamboo  rod.  In  preparations  made  by  staining 
blood  or  other  animal  juices  the  bacilli  often  appear  sur- 
rounded by  transparent  capsules.  Such  are  not  found  in 
specimens  made  from  artificial  cultures. 


' 


'. 


.. 

-y*;# 


,,;   <T, 
I        ' 


\        *» 


Fig.    124. — Bacillus  anthracis,  stained  to  show  the  spores.      X    1000 
(Frankel  and  Pfeiffer). 

Sporulation. — The  formation  of  endospores  is  prolific: 
each  spore  has  a  distinct  oval  shape,  is  transparent,  situated 
at  the  center  of  the  bacillus  in  which  it  occurs.  It  does  not 
alter  the  contour  of  the  bacillus.  The  spores  are  formed 
only  in  the  presence  of  oxygen  upon  the  surfaces  of  the 
culture-media.  When  a  spore  is  placed  under  conditions 
favorable  to  its  development,  it  increases  in  length  and 
ruptures  at  the  end,  from  which  the  new  bacillus  escapes. 
The  spores  of  the  anthrax  bacillus,  being  large  and  readily 
obtainable,  form  excellent  subjects  for  the  study  of  spore- 
formation  and  germination,  for  the  study  of  the  action  of 
germicides  and  antiseptics,  and  for  staining. 


Cultivation  403 

Motility. — The  bacilli  are  not  motile  and  have  no  flagella. 

Staining. — They  stain  well  with  ordinary  solutions  of  the 
anilin  dyes,  and  can  be  beautifully  demonstrated  in  the 
tissues  by  Gram's  method  and  by  Weigert's  modification  of 
it.  Picrocarmin,  followed  by  Gram's  stain,  gives  a  beau- 
tiful, clear  picture.  The  spores  can  be  stained  by  any  of  the 
special  methods  for  staining  spores  (q.  v.). 

Isolation. — The  bacillus  of  anthrax  is  one  of  the  easiest 
organisms  to  secure  in  pure  culture  from  the  tissues  and 
excreta  of  diseased  animals.  Its  luxurious  vegetation,  the 
typical  appearance  of  its  colonies,  and  its  infectivity  for  the 
laboratory  animals  combine  to  make  possible  its  isolation 
either  by  direct  cultivation  from  the  tissues,  by  the  plate 
method,  or  by  the  inoculation  into  animals  and  recovery 
of  the  micro-organisms  from  their  blood. 


Fig.    125. — Bacillus  anthracis;  colony  upon  a  gelatin  plate.     X  100 
(Frankel  and  Pfeiffer). 

Cultivation. — Colonies. — Upon  the  surface  of  a  gelatin 
plate  the  bacillus  forms  beautiful  and  highly  characteristic 
colonies  (Fig.  125).  To  the  naked  eye  they  appear  first  as 
minute  round,  grayish-white  dots  upon  the  surface.  They 
early  begin  liquefaction  of  the  gelatin,  which  progresses 
rapidly  as  they  increase  in  size.  Under  the  microscope  the 
smallest  colonies  are  egg-shaped,  slightly  brown  and  granular. 
They  do  not  attain  their  full  development  except  upon  the 


404 


Anthrax 


Fig.  126.— Bacillus  an- 
thracis ;  gelatin  stab  cul- 
ture, showing  character- 
istic growth  with  com- 
mencing liquefaction  and 
cupping  (from  evapora- 
tion) at  the  surface  of 
the  medium  (Curtis). 


surface  of  the  medium,  where  they 
spread  out  into  flat,  irregular, 
transparent  tufts  like  curled  wool. 
From  a  tangled  center  large  num- 
bers of  curls,  made  up  of  parallel 
threads  of  bacilli,  extend  upon  the 
gelatin.  As  soon  as  the  colony  at- 
tains to  any  considerable  size  lique- 
faction becomes  rapid.  Beautiful 
adhesion  preparations  can  be  made 
if  a  perfectly  clean  cover-glass  be 
passed  once  through  a  flame  and 
laid  carefully  upon  the  gelatin,  the 
colonies  being  picked  up  entire  as 
the  glass  is  carefully  removed.  Such 
a  specimen  can  be  dried,  fixed,  and 
stained  in  the  same  manner  as  an 
ordinary  cover-glass  preparation. 

Gelatin  Punctures. — In  gelatin 
puncture  cultures  the  growth  is 
even  more  characteristic  than  are 
the  colonies.  The  bacilli  begin  to 
grow  along  the  entire  track  of  the 
wire,  but  develop  most  luxuriantly 
at  the  surface,  where  oxygen  is 
plentiful  and  where  a  distinct 
shaggy  pellicle  is  formed.  From  the 
deeper  growth,  fine  filaments  extend 
from  the  puncture  into  the  sur- 
rounding gelatin,  with  a  beautiful 
arborescent  effect  (Fig.  126). 

Liquefaction  progresses  from 
above  downward  until  ultimately 
the  entire  gelatin  is  fluid  and  the 
growth  sediments. 

Agar-agar.  —  Upon  agar-agar 
characteristic  appearances  are  few. 
The  growth  takes  place  along  the 
line  of  inoculation,  forming  a 
grayish-white,  translucent,  slightly 
wrinkled  layer  with  irregular  edges, 
from  which  curls  of  bacillary  threads 
extend  upon  the  medium.  When 


Metabolic  Products  405 

the  culture  is  old,  the  agar-agar  usually  becomes  brown  in 
color.     Spore  formation  is  luxuriant. 

Bouillon. — In  bouillon  the  anthrax  bacillus,  because  of  its 
marked  affinity  for  oxygen,  grows  chiefly  upon  the  surface, 
where  a  thick  felt-like  pellicle  forms.  From  this,  fuzzy  ex- 
tensions descend  into  the  clear  bouillon  below.  After  a  few 
days  some  wooly  aggregations  can  be  seen  in  the  bottom  of 
the  tube.  In  the  course  of  time  the  growth  ceases  and  the 
surface  pellicle  sinks.  If,  by  shaking,  it  is  caused  to  sink 
prematurely,  a  new,  similar  surface  growth  takes  its  place. 
Spore  formation  is  rapid  at  the  surface. 

Potato. — Upon  the  potato  the  growth  is  white,  creamy, 
and  rather  dry.  Sporulation  is  marked. 

Blood-serum. — Blood-serum  cultures  lack  characteristic 
peculiarities;  the  culture-medium  is  slowly  liquefied. 

Milk. — The  anthrax  bacillus  grows  well  in  milk,  which 
it  coagulates  and  acidulates.  Later  the  coagulum  is  pep- 
tonized  and  dissolved,  leaving  a  clear  whey.  The  reaction 
is  not  altered.  Iwanow*  found  that  the  organism  forms 
acetic,  formic,  and  caproic  acids. 

Thermic  Sensitivity. — The  bacillus  grows  between  the 
extremes  of  12°  and  45°  C.,  best  at  37°  C.  The  exposure  of 
the  organism  to  the  temperature  of  42°  to  43°  C.  slowly  di- 
minishes its  virulence. 

When  dried  upon  threads,  the  spores  retain  their  vital- 
ity for  years,  and  are  highly  resistant  to  heat  .and  disinfec- 
tants. The  spores  of  anthrax  are  killed  by  five  minutes' 
exposure  to  100°  C.  It  is  said  by  some  that  spores  sub- 
jected to  5  per  cent,  carbolic  acid  can  subsequently  ger- 
minate when  introduced  into  susceptible  animals,  their 
resistance  to  this  strength  carbolic  solution  being  so  great 
that  they  are  not  destroyed  by  it  under  twenty-four  hours. 
They  are  killed  in  a  short  time  by  exposure  to  i  :  1000 
bichlorid  of  mercury  solution. 

Metabolic  Products. — The  anthrax  bacillus  produces  a 
curdling  ferment.  It  produces  no  important  change  of 
reaction  in  the  medium  in  which  it  grows,  and  generates  no 
indol.  Its  proteolytic  enzyme  is  active,  digesting  both 
casein  and  fibrin. 

It  is  doubtful  whether  the  anthrax  bacillus  produces  any 
important  toxic  substance.     Hoffaf  isolated  a  basic  sub- 
*  "Ann.  de  1'Inst.  Pasteur,"  1892. 
t  "Ueber  die  Natur.  des  Milzbrandgifts,"  Wiesbaden,  1886. 


406 


Anthrax 


Fig.  127. — Bacillus  an- 
thracis;  glycerin  agar- 
agar  culture  (Curtis). 


stance  from  anthrax  cultures  and 
called  it  anthracin;  Hankin  and  Wes- 
brook,*  an  albumose  fatal  in  large 
doses  and  immunizing  in  small  ones. 
Brieger  and  Frankel  f  isolated  a  tox- 
albumin  from  the  tissues  of  animals 
dead  of  anthrax.  Martin  J  separated 
protalbumose,  deuteroalbumose, 
peptone,  an  alkaloid,  leucin,  and 
tyrosin.  The  albumoses  were  not 
very  poisonous,  but  the  alkaloid  was 
capable  of  producing  death  after  the 
development  of  somnolence.  The 
animals  were  edematous.  Marmier  § 
isolated  a  toxin  of  non-albuminous 
nature  and  immunizing  power.  Con- 
radi  ||  in  an  elaborate  research  failed 
to  find  that  the  anthrax  bacillus  pro- 
duces any  soluble  extracellular  or  in- 
tracellular  poison  capable  of  affect- 
ing susceptible  animals,  and  con- 
cludes that  it  is  highly  improbable 
that  the  anthrax  bacillus  produces 
any  toxic  substance  at  all. 

Pathogenesis. — Avenues  of  In- 
fection.— Infection  usually  takes 
place  through  the  respiratory  tract, 
through  the  alimentary  canal,  or 
through  wounds.  It  may  take 
place  through  the  placenta. 

When  the  bacilli  are  taken  into 
the  stomach  they  are  probably  de- 
stroyed by  the  acid  gastric  juice. 
The  spores,  however,  are  able  to  en- 
dure the  acid  gastric  juice,  and  pass 
into  the  intestine,  where  the  suitable 
alkalinity  enables  them  to  develop 
into  bacilli,  surround  the  villi  with 


*"Ann.  de  1'Inst.  Pasteur,"  1892,  No.  9. 
f'Ueber  Ptomaine,"  Berlin,  1885-1886. 
t  "Proceedings  of  the  Royal  Society,"  May  22,  1890. 
§  "Ann.  de.l'Inst.  Pasteur,"  1895,  p.  533. 
||"Zeitschrift  fur  Hygiene,"  June  14,  1899. 


Pathogenesis  407 

thick  networks  of  bacillary  threads,  separate  the  covering 
epithelial  cells,  enter  the  lymphatics,  and  then  the  blood, 
from  which  a  general  infection  occurs. 

The  bacillus  frequently  enters  the  body  through  wounds, 
cuts,  scratches,  and  perhaps  occasionally  fly-bites.  Under 
these  conditions  the  organisms  at  once  find  themselves  in 
the  lymphatics  or  capillaries,  and  may  cause  immediate 
general  infection.  In  human  beings  a  "  malignant  pustule  " 
is  apt  to  follow  local  infection,  and  may  recover  or  ulti- 
mately cause  death  by  general  infection.  Those  whose 
occupations  bring  them  in  contact  with  the  skins  and 
hair  from  animals  dead  of  anthrax  are  liable  to  the  infection. 

Anthrax  in  cattle  probably  results  from  the  inhalation 
or  ingestion  of  the  spores  of  the  bacilli  from  the  pasture. 
From  the  work  of  Nuttall  *  it  is  pretty  clear  that  flies 
play  little  part  in  the  transmission  of  the  disease.  Inter- 
esting discussions  arose  concerning  the  infection  of  the 
pastures.  It  was  argued  that,  the  bacilli  being  inclosed  in 
the  tissues  of  the  diseased  animals,  infection  of  the  pasture 
must  depend  upon  the  distribution  of  the  germs  from  buried 
cadavers,  either  through  the  activity  of  earthworms,  which 
ate  of  the  earth  surrounding  the  corpse  and  deposited  the 
spores  in  their  excrement  (Pasteur),  or  to  currents  of  mois- 
ture in  the  soil.  Koch  seems,  however,  to  have  demon- 
strated the  fallacy  of  both  theories  by  showing  that  the 
conditions  under  which  the  bacilli  find  themselves  in  buried 
cadavers  are  opposed  to  fructification  or  sporulation,  and 
that  in  all  probability  the  bacteria  suffer  the  same  fate  as 
the  cells  of  the  buried  animals,  and  disintegrate,  especially 
if  the  animal  be  buried  at  a  depth  of  two  or  three  meters. 

Frankel  points  out  particularly  that  no  infection  of  the 
soil  by  the  dead  animal  could  be  worse  than  the  pollution 
of  its  surface  by  the  bloody  stools  and  urine,  rich  in  bacilli, 
discharged  upon  it  by  the  animal  before  death,  and  that 
it  is  the  live,  and  not  the  dead,  animals  that  are  to  be  blamed 
for  the  infection. 

Lesions. — The  disease  as  seen  in  the  laboratory  is 
accompanied  by  few  marked  lesions.  The  ordinary  ex- 
perimental inoculation  is  made  by  cutting  away  a  little 
of  the  hair  from  the  abdomen  of  a  guinea-pig  or  rabbit,  or 
at  the  root  of  a  mouse's  tail,  making  a  little  subcutaneous 
pocket  by  a  snip  with  sterile  scissors,  and  introducing  the 
*  "Johns  Hopkins  Hospital  Reports,"  1899. 


408 


Anthrax 


spores  or  bacilli  with  a  heavy  platinum  wire,  the  end  of  which 
is  flattened,  pointed,  and  perforated.  An  animal  inoculated 
in  this  way  dies,  according  to  the  species,  in  from  twenty 
four  hours  to  three  days,  suffering  from  weakness,  fever, 
loss  of  appetite,  and  a  bloody  discharge  from  nose  and 
bowels.  There  is  much  subcutaneous  gelatinous  edema 
near  the  inoculation  wound.  The  abdominal  viscera  are 
injected  and  congested.  The  spleen  is  enlarged,  dark  in 
color,  and  of  mushy  consistence.  The  liver  is  also  some- 
what enlarged.  The  lungs  are  usually  slightly  congested. 


r 


Fig.  128. — Anthrax  bacilli  in  glomeruli  of  kidney. 


When  organs  which  present  no  appreciable  changes  to  the 
naked  eye  are  subjected  to  a  microscopic  examination,  the 
appropriate  staining  methods  show  the  capillary  and 
lymphatic  systems  to  be  almost  universally  occupied  by 
bacilli,  which  extend  throughout  their  meshworks  in  long 
threads.  Most  beautiful  bacillary  threads  can  be  found  in 
the  glomeruli  of  the  kidney  and  in  the  minute  capillaries 
of  the  intestinal  villi.  In  the  larger  vessels,  where  the 
blood-stream  is  rapid,  no  opportunity  is  afforded  for  the 
formation  of  the  threads,  and  the  bacteria  are  relatively 


Vaccination  409 

few.  so  that  the  burden  of  bacillary  obstruction  is  borne  by 
the  minute  vessels.  The  condition  is  thus  a  pure  bacteremia. 

Death  from  anthrax  seems  to  depend  essentially  upon 
the  obstruction  of  the  circulation  by  the  multitudes  of 
bacilli  in  the  capillaries,  upon  the  appropriation  of  the 
oxygen  destined  to  support  the  tissues,  by  the  bacilli, 
leaving  the  tissues  to  be  poisoned  by  the  carbon*  dioxid, 
rather  than  upon  intoxication  by  metabolic  products  of 
bacillary  growth. 

Vaccination. — Pasteur  *  early  realized  the  importance 
of  some  practical  measure  for  the  protective  vaccination 
of  cattle  against  the  disease,  and  devoted  himself  to  inves- 
tigating the  problem.  He  found  that  the  inoculation  of 
attenuated  bacilli  into  cows  and  sheep,  and  their  subse- 
quent reinoculation  with  mildly  virulent  bacilli,  afforded 
them  immunity  against  highly  virulent  organisms.  Loffler, 
Koch,  and  Gaffky,  however,  found  that  these  immunized 
animals  were  not  absolutely  protected  against  intestinal 
anthrax. 

The  means  of  diminishing  the  virulence  of  the  anthrax 
bacillus  are  numerous.  Toussaint  f  first  produced  im- 
munity in  animals  by  injecting  them  with  sterile  cultures  of 
the  bacillus,  and  found  that  the  addition  of  i  per  cent,  of 
carbolic  acid  to  blood  of  animals  dead  of  anthrax  destroyed 
the  virulence  of  the  bacilli ;  Chamberland  J  and  Roux  found 
the  virulence  destroyed  when  0.1-0.2  per  cent,  of  bichro- 
mate of  potassium  was  added  to  the  culture  medium; 
Chauveau  used  atmospheric  pressure  to  the  extent  of  six  to 
eight  atmospheres  and  found  the  virulence  diminished; 
Arloing  §  found  that  direct  sunlight  operated  similarly; 
Lubarsch,  that  the  inoculation  of  the  bacilli  into  immune 
animals,  such  as  the  frog,  and  their  subsequent  recovery 
from  its  blood,  diminishes  the  virulence  of  the  bacilli  mark- 
edly. 

The  protective  inoculations  prepared  by  Pasteur  consisted 
of  two  cultures  of  increasing  virulence,  to  be  employed  one 
after  the  other,  rendering  the  vaccinated  animals  more  and 
more  immune.  The  cultures  were  prepared,  that  is,  at- 

*  "Rec.  de  Med.  vet.,"  Paris,  1879,  p.  193. 

t  "  Compte-rendu  Acad.  des  Sci.  de  Paris,"  xci,  1880,  p.  135. 

t  "Ann.  de  1'Inst.  Pasteur,"  1894,  p.  161. 

\  "Compte-rendu  de  1'Acad.  des  Sci.,"  Paris,  1892,  cxiv,  p.  1521. 


410  Anthrax 

tenuated  by  cultivation  at  42°  C.  for  a  sufficient  length  of 
time,  the  bacilli  forming  no  spores  and  gradually  losing 
their  virulence  at  this  temperature.  The  first  vaccine  was  kept 
from  fifteen  to  twenty  days  at  42°  C.  It  killed  mice  and 
guinea-pigs  one  day  old,  but  was  without  action  on  guinea- 
pigs  of  adult  size.  The  second  vaccine  only  remained  at 
the  temperature  of  42°  C.  for  from  ten  to  twelve  days 
and  killed  mice,  guinea-pigs,  and  occasionally  rabbits. 

The  second  vaccine  is  administered  from  two  to  three 
weeks  after  the  first  is  given,  by  hypodermic  injection  into 
the  tissues  of  the  neck  or  flank.  Of  each  broth  culture 
about  i  c.c.  is  administered.  The  animals  frequently  be- 
come ill. 

Pasteur  demonstrated  the  value  of  his  method  in  1881  at 
Pouilly-le-Fort,  in  a  manner  so  convincing  to  the  entire 
world  that  it  was  immediately  put  into  practice  in  France. 
Roger*  says  that  between  1882  and  1894  there  were 
1,788,677  sheep  vaccinated,  with  a  mortality  of  0.94  per 
cent.,  the  previous  death-rate  having  been  10  per  cent. 
There  were  also  200,962  cattle  vaccinated,  with  a  reduction 
of  the  death-rate  from  5  per  cent,  to  0.34  per  cent. 

Chamberland  has  shown  that  protective  inoculation  by 
Pasteur's  method  has  diminished  the  death-rate  from  10 
per  cent,  for  sheep  and  5  per  cent,  for  cattle  to  about  0.94 
per  cent,  for  sheep  and  0.34  per  cent,  for  cattle,  so  that 
the  utility  of  the  method  is  scarcely  questionable.  The 
method  has  been  less  successful  elsewhere  than  in  France, 
and  has  sometimes  caused  the  death  of  the  animals  to  be 
protected. 

Protection  against  anthrax  can  be  afforded  in  other  ways. 
Hiippe  found  that  the  simultaneous  inoculation  of  bacteria 
not  at  all  related  to  anthrax  will  sometimes  cause  the 
animal  to  recover.  Hankin  found  in  the  cultures  chemic 
substances,  especially  an  albuminose,  that  exerted  a  pro- 
tective influence.  Rettgerf  prepared  ' '  prodigiosus  powder ' ' 
from  potato  cultures  of  B.  prodigiosus,  which  when  injected 
into  guinea-pigs  during  experimental  anthrax  infection  pro- 
longed life  or  induced  recovery. 

Serum  Therapy. — In  1890  Ogata  and  Jasuhara  showed 
that  experiment  animals  convalescent  from  anthrax  pos- 
sessed an  antitoxic  substance  in  the  blood  of  such  strength 

*"Les  Maladies  Infectieuse,"  n,  p.  1489. 

f  "Jour,  of  Infectious  Diseases,"  vol.  n,  No.  4,  p.  562,  Nov.  25,  1905. 


Bacilli  Resembling  the  Anthrax  Bacillus       411 

that  i  :  800  parts  per  body-weight  of  dog's  serum  contain- 
ing the  antitoxin  would  protect  a  mouse.  Similar  results 
have  been  attained  by  Marchoux.  *  Serum  therapy  in 
anthrax  is,  however,  of  no  practical  importance  either  for 
prophylaxis  or  treatment,  as  vaccinating  the  animals  is 
far  cheaper  and  more  satisfactory. 

Bacteriologic  Diagnosis. — When  it  is  desired  to  have 
a  bacteriologic  diagnosis  of  anthrax  made  where  no  labora- 
tory facilities  are  at  hand,  an  ear  of  the  dead  animal  can 
be  inclosed  in  a  bottle  or  fruit  jar  and  sent  to  the  nearest 
laboratory  where  diagnosis  can  be  made.  The  ear  contains 
so  little  readily  decomposable  tissue,  that  it  keeps  fairly  well, 
drying  rather  than  rotting.  It  contains  enough  blood  to 
enable  a  bacteriologist  to  make  a  successful  examination. 

Sanitation. — As  every  animal  affected  with  anthrax  is  a 
menace  to  the  community  in  which  it  lives, — to  the  men  who 
handle  it  as  well  as  the  animals  who  browse  beside  it, — 
such  animals  should  be  killed  as  soon  as  the  diagnosis  is 
made,  and,  together  with  the  hair  and  skin,  be  burned, 
or  if  this  be  impracticable,  Frankel  recommends  that  they 
be  buried  to  a  depth  of  at  least  1^-2  meters,  so  that  the 
sporulation  of  the  bacilli  is  made  impossible.  The  dejecta 
should  also  be  carefully  disinfected  with  5  per  cent,  carbolic 
acid  solution. 


BACILLI  RESEMBLING  THE  ANTHRAX  BACILLUS. 
Bacilli  presenting  the  'morphologic  and  cultural  char- 
acteristics of  the  anthrax  bacillus,  but  devoid  of  any  disease- 
producing  power,  are  occasionally  observed.  Of  these, 
Bacillus  anthracoides  of  Hiippe  and  Wood,f  Bacillus  anthra- 
cis  similis  of  McFarland,J  and  Bacillus  pseudoanthracis§ 
have  been  given  special  names.  What  relationship  they 
bear  to  the  anthrax  bacillus  is  uncertain.  They  may  be 
entirely  different  organisms,  or  they  may  be  individuals 
whose  virulence  has  been  completely  lost  through  un- 
favorable environment. 

*  "Ann.  de  1'Inst.  Pasteur,"  November,    1895,  t.  ix,   No.   11,  pp. 
50-75. 

f  "Berliner  klin.  Wochenschrift,"  1889.  16. 

t  "Centralbl.  f.  Bakt.,"  vol.  xxiv,  No.  26,  p.  556. 

\  "Hygienische  Rundschau,"  1894,  No.  8. 


CHAPTER   V. 


HYDROPHOBIA,  LYSSA,  OR  RABIES. 

HYDROPHOBIA,  lyssa,  or  rabies  is  a  specific  infectious 
toxic  disease  to  which  dogs,  wolves,  skunks,  and  cats  are 
highly  susceptible,  and  which,  through  their  saliva,  can  be 
communicated  to  men,  horses,  cows,  and  other  animals. 
The  means  of  communication  is  almost  invariably  a  bite, 
hence  the  specific  organism  must  be  present  in  the  saliva. 

The  infected  animals  manifest  no  symptoms  during  a  vary- 
ing incubation  period  in  which  the  wound  heals  kindly. 
This  period  may  be  of  twelve  months'  duration,  but  in  rare 
cases  may  be  only  a  few  days.  The  average  duration  of  the 
period  of  incubation  is  about  six  weeks. 

Toward  the  close  of  the  incubation  period  an  observable 
alteration  occurs  in  the  wound,  which  becomes  reddened, 
may  suppurate,  and  is  painful.  The  victim  has  a  sensation 
of  horrible  dread,  which  passes  into  wild  excitement,  with 
paralysis  of  the  pharyngeal  muscles  and  inability  to  swallow. 
The  wild  delirium  ends  in  a  final  stage  of  convulsion  or 
palsy.  The  convulsions  are  tonic,  rarely  clonic,  and  finally 
cause  death  by  interfering  with  respiration. 

During  the  convulsive  period  much  difficulty  is  experi- 
enced in  swallowing  liquids,  and  it  is  supposed  that  the 
popular  term  "  hydrophobia  "  arose  from  the  reluctance  of 
the  diseased  to  take  water  because  of  painful  spasms  caused 
by  the  attempt. 

Pasteur*  and  his  co-workers,  Chamber-land  and  Roux,f 
found  that  in  animals  that  die  of  rabies  the  salivary  glands, 

*  "Compte-rendu  Acad.  des  Sciences,"  Paris,  1889,  cvm,  p.  1228. 
t  Ibid.,  Oct.  26,  1885. 

412 


Pasteur's  Treatment  413 

the  pancreas,  and  the  nervous  system  contain  the  infection, 
and  are  more  appropriate  for  experimental  purposes  than 
the  saliva,  which  is  invariably  contaminated  with  accidental 
pathogenic  bacteria. 

The  introduction  of  a  fragment  of  the  medulla  oblongata 
of  a  dog  dead  of  rabies,  beneath  the  dura  mater  of  a  rabbit, 
causes  the  development  of  typical  rabies  in  the  rabbit  in 
about  six  days.  It  is  only  by  such  an  inoculation  that  a  posi- 
tive diagnosis  of  the  disease  can  be  made.  The  operation 
must  be  performed  with  the  greatest  care  in  order  to  avoid 
septic  infection  with  meningitis.  The  technic  is  simple,  a 
small  trephine  for  opening  the  rabbit's  skull  being  obtainable 
from  the  dealers,  though  in  its  absence  the  thin  bone  of  the 
cranial  cavity  may  be  cut  with  a  heavy  scalpel.  The  material 
to  be  inoculated  should  be  crushed  to  a  fine  pulp  in  sterile 
physiologic  salt  solution,  and  introduced  beneath  the  dura 
with  a  hypodermic  syringe.  The  tissue  of  the  medulla  of  a 
rabid  rabbit  introduced  beneath  the  dura  mater  of  a  second 
rabbit  produces  a  more  violent  form  of  the  disease  in  a  shorter 
time,  and  by  frequently  repeated  implantations  Pasteur 
found  that  an  extremely  virulent  material  could  be  ob- 
tained. 

Pasteur  observed  that  the  virulence  of  the  poison  was  less 
in  animals  that  had  been  dead  for  some  time  than  in  those 
just  killed,  and  by  experiment  found  that  when  the  ner- 
vous system  of  an  infected  rabbit  was  dried  in  a  sterile  at- 
mosphere its  virulence  attenuated  in  proportion  to  the 
length  of  time  it  was  kept.  A  method  of  attenuating  the 
virulence  was  thus  suggested  to  Pasteur,  and  the  idea  of 
using  it  as  a  protective  vaccination  soon  followed.  After 
careful  experimentation  he  found  that  by  inoculating  a 
dog  with  much  attenuated,  then  with  less  attenuated,  then 
with  moderately  strong,  and  finally  with  a  strong,  virus,  it 
developed  an  immunity  that  enabled  it  to  resist  infection 
with  an  amount  of  virulent  material  that  would  certainly 
kill  an  unprotected  dog. 

It  is  remarkable  that  this  theory,  based  upon  limited  accu- 
rate biologic  knowledge,  and  upon  experience  with  very  few 
bacteria,  should  find  absolute  confirmation  as  our  knowledge 
of  immunity,  toxins,  and  antitoxins  progressed.  Pasteur 
introduced  the  unknown  poison-producers,  attenuated  by 
drying,  and  capable  of  generating  only  a  little  poison,  accus- 


414  Hydrophobia,  Lyssa,  or  Rabies 

tomed  the  animal  first  to  them  and  then  to  stronger  and 
stronger  ones  until  immunity  was  established. 

For  the  treatment  of  infected  cases  exactly  the  same  method 
is  followed  as  for  the  production  of  immunity.  Indeed,  the 
treatment  of  a  patient  bitten  by  a  rabid  animal  is  simply  the 
production  of  immunity  during  the  prolonged  incubation 
period  of  the  affection,  so  that  the  disease  may  not  develop. 
The  patient,  to  be  successfully  treated,  must  come  under 
observation  early. 

Method.— To  protect  human  beings  from  the  development 
of  hydrophobia  after  they  have  been  bitten  by  rabid  animals, 
it  is  necessary  to  use  material  of  standard  or  known  virulence. 
This  can  be  prepared,  according  to  the  directions  of  Hogyes,* 
by  the  passage  of  virus  from  a  rabid  animal  through  from 
21  to  30  rabbits.  For  this  purpose  some  of  the  hippocampal 
tissue  of  the  dog  is  made  into  an  emulsion  with  sterile  salt 
solution  and  injected  subcutaneously  into  a  rabbit.  As  soon 
as  this  animal  dies,  its  spinal  cord  is  removed,  a  similar  emul- 
sion made  with  a  fragment  of  it,  and  a  second  rabbit  inoculated, 
and  so  on  through  the  series  until  a  standard  virulence  is 
attained  and  the  virus  is  said  to  be  "  fixed."  It  has  a  much 
higher  degree  of  virulence  than  the  "  street  virus  "  taken  from 
the  rabid  dog,  but  its  virulence  does  not  vary.  In  most 
laboratories  the  "fixed  virus"  is  obtained  from  other  labora- 
tories and  kept  passing  through  rabbits.  In  this  manner 
uniformity  of  dosage  and  virulence  is  most  easily  main- 
tained. 

The  technic  of  obtaining  the  rabbit's  cord  given  by  Oshidaf 
is  the  one  now  generally  employed.  As  given  by  Stimson,  J  it 
is  performed  at  the  Hygienic  Laboratory  as  follows:  "The 
rabbit,  when  completely  paralyzed,  is  killed  with  chloroform 
and  nailed  to  a  board,  back  uppermost,  and  thoroughly 
wetted  down  with  an  aseptic  solution  (i  per  cent,  trikresol). 
An  incision  is  made  through  the  skin  from  the  forehead  nearly 
to  the  tail  and  the  skin  laid  back  on  each  side,  the  ears 
being  cut  close  to  the  head.  An  area  i  inch  wide  is  seared 
with  a  hot  iron  around  the  occiput  and  nuchal  region  and  ear 
openings.  The  skin  is  then  transversely  divided  in  the  center 

*  See  Kraus  and  Levaditi,  "Handbuch  der  Immunitatsforschung,"  i. 
t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1901,  xxix,  Orig.,  988. 
t"  Facts  and  Problems  of  Rabies,"  Hygienic  Laboratory  Bulletin, 
No.  65,  June,  1910,  Washington,  D.  C. 


Method 

of  the  seared  areas  by  means  of  bone-cutting  forceps.  The 
neck  is  dissected  loose  from  the  skin  and  a  large  square  of 
sterile  gauze  is  inserted  beneath  it.  The  lumbar  region  is 
dissected  up  for  a  few  inches  and  a  similar  piece  of  gauze 
placed  beneath  it.  Then  a  piece  of  telegraph  wire  about  14 
inches  long,  bent  into  a  handle  at  one  end  and  having  a  small 
wisp  of  cotton  twisted  about  the  other  end,  is  used  to  push  the 
cord  out  of  its  canal.  The  spine  is  steadied  by  a  pair  of  lion- 
jawed  forceps. 


Fig.  129. — Removal  of  the  spinal  cord  from  a  rabbit  (Stimson,  Bull.  No. 
65,  Hygienic  Laboratory). 


An  assistant  catches  the  cord  with  forceps  as  it  emerges 
from  the  cervical  opening  and  lifts  it  out.  The  spinal  nerves 
are  torn  off  during  this  procedure,  and  the  membranes  stripped 
off,  leaving  a  clean  sterile  cord.  A  silk  ligature  with  one  long 
end  is  placed  around  the  upper  end,  and  another,  just  below  the 
middle  of  the  cord,  which  is  then  cut  into  two  pieces  just  above 
the  lower  ligature.  A  small  piece  is  cut  off  of  the  upper  end  of 
the  upper  portion  and  placed  in  a  tube  of  bouillon,  which  is 
incubated  as  a  test  for  sterility.  The  cords  are  hung  in  the 
drying  bottle. 

The  cords  thus  secured  are  used  for  perpetuating  the 


4i 6  Hydrophobia,  Lyssa,  or  Rabies 

"  fixed  virus  "  by  inoculating  other  rabbits,  and  after  the 
virus  has  become  "  fixed  "  are  also  used  for  treating  the 
bitten  patient. 

For  this  purpose,  however,  Pasteur  attenuated  the  virus 
by  drying  the  cords,  placing  them  in  a  bottle  containing 
caustic  potash. 

The  longer  the  cord  dries,  the  more  the  virulence  of  the 
micro-organisms  attenuates,  and  as  in  beginning  the  immuni- 


Fig.  130. — Method  of  drying  the  spinal  cord  of  a  rabbit  for  the  purpose  of 
attenuation  (Stimson,  Bull.  No.  65,  Hygienic  Laboratory). 


zation  of  human  beings  it  is  essential  to  use  very  attenuated 
material,  the  first  cord  used  to  furnish  inoculation  material 
must  have  dried  about  ten  days. 

The  cord,  when  it  has  reached  the  necessary  attenuation, 
is  transferred,  and  i  cm.  of  it  is  emulsified  with  3  c.c.  of  sterile 
0.8  per  cent,  salt  solution.  There  can  be  no  absolute  ac- 
curacy of  dosage.  The  injection  material  made  in  the 
laboratory  under  strict  aseptic  precautions  can  be  used  with 
perfect  safety  for  many  hours  subsequently  if  kept  cold,  and 


Pasteur's  Original  Schemata 


can  be  packed  in  ice  and  sent  by  express  to  the  physician  to 
use  at  the  home,  of  his  patients. 

As  the  transfer  of  the  cord  to  glycerin  preserves  the  viru- 
lence at  whatever  degree  it  had  when  so  transferred,  it  is  now 
customary  to  keep  on  hand,  in  glycerin,  in  the  laboratory, 
spinal  cords  of  rabbits  dried  one,  two,  three,  four  days,  and 
so  on  through  the  whole  series. 

This  makes  it  possible  to  keep  on  hand,  always  available, 
material  for  furnishing  all  the  vaccines  at  any  time,  indepen- 
dently of  the  experimental  rabbits,  and  also  makes  it  possible 
for  one  rabbit  cord  to  serve  several  cases.  The  cords  in 
glycerin,  if  kept  in  the  cold,  do  not  change  in  virulence  for 
several  weeks. 

The  following  table  shows  Pasteur's  original  schemata: 


PASTEUR'S  ORIGINAL  SCHEMATA    (Marx). 


Light  schema. 

Intense  schema. 

Day  of  treatment. 

! 

Age  of    Amount  of 
dried          injected 
cord.    |    emulsion. 

Day  of  treatment. 

Age  of 
dried 
cord. 

Amount  of 
injected 
emulsion. 

First. 

Days  . 

l«4 
1i3 

" 

h 

I;'! 

{^ 

5 

4 
3 

5 
5 
4 
4 
3 
3 
5 
4 
3 

c.c. 

3 
3 
3 
3 
3 
3 
3 
3 

First  

Days. 

M 
»3 

12 
II 
IO 

9 

8 

I 

\   6 
5 

1 

4 
3 
3 

4 
3 
5 
4 
3 

c.c. 

3 
3 
3 
3 
3 
3 
3 
3 

Second 

Second 

Third 

Fourth  
Fifth  

Third  

Sixth. 

Fourth  
Fifth 

Eighth  
Ninth 

Sixth  
Seventh 

Tenth  
Eleventh  
Twelfth 

Eighth  .  . 

Ninth  
Tenth 

Thirteenth  
Fourteenth  
Fifteenth  
Sixteenth  
Seventeenth  
Eighteenth 

Eleventh... 
Twelfth  
Thirteenth 

Fourteenth  

Fifteenth  
Sixteenth 

Seventeenth  
Eighteenth  
Nineteenth. 

Twentieth  

Twenty-first.  .  . 

(From  Bulletin  No.  65,  Hygienic  Laboratory,  June,  1910,  U.  S.  Public 

Health  and  Marine-Hospital  Service.) 
27 


4i8 


Hydrophobia,  Lyssa,  or  Rabies 


The  system  of  treatment  at  present  used  at  the  Hygienic 
Laboratory  is  shown  in  the  following  tables: 

SCHEME  FOR  MILD   TREATMENT. 


Amount  injected. 

Amount  injected. 

Day. 

Cord. 

Five 

One 

Day. 

Cord. 

Five 

One 

Adult. 

to  ten 

to  five 

Adult,  to  ten 

to  five 

years. 

years. 

years. 

years. 

Injections. 

c.c. 

c.c. 

c.c. 

Injections. 

c.c. 

c.c. 

c.c. 

j 

8—7—6=3 

2.5 

2-5 

2.O 

12 

2  

5—4=2 

2-5 

2-5 

i-5 

13   

4= 

•5 

•5 

-5 

3  

4  —  3—2 

2-5 

2-5 

2.O 

14 

4  

5= 

2-5 

2.5 

2.5 

1C 

5  
6 

4= 

2.5 

2.5 

2-5 

ii::::::::: 

2  = 

•5 

.O 

•5 

3= 

2.5 

2.5 

2.0 

IS::::::::: 

4= 

2-5 

•5 

•5 

2  — 

9  •. 

2  = 

2.5 

2  O 

i  5 

20 

10  

5= 

2-5 

2.5 

2-S 

21  

2= 

2-5 

2-5 

.O 

ii  

5=r 

2.5 

2.5 

2-5 

SCHEME  FOR  INTENSIVE  TREATMENT. 


Day. 

Cord. 

Amount  injected. 

Day. 

Cord. 

Amount  injected. 

Adult. 

Five 
to   ten 
years. 

One 

to  five 
years. 

Adult. 

Five 
to  ten 
years. 

One 
to  five 
years. 

i  

Injections. 
8—7—6=3 

c.c. 

2-5 

c.c. 

2-5 

c.c. 
2.5 

il2  . 

Injections. 

3= 
3= 

2  = 
2  = 

4= 

3  = 

2  = 

3= 

2  = 
1  = 

c.c. 

2-5 
•5 
•5 
•5 
•5 
•  5 
•5 
•5 
•5 
-5 

c.c. 
2-5 

2.5 

•  5 
•  5 
-5 
•  5 
•5 
•5 
2-5 

2-5 

c.c. 

2.0 
2.0 

2.O 
2.0 
2-5 
2-5 
2.0 
2.O 
2-5 
2.0 

2                      ... 

4—3= 
5—4= 
3= 
3= 

2  = 
2  = 

5= 
4= 
4= 

2.5 
2.5 

2-5 

2.5 
2.5 
2.5 

2.5 

2.5 

2.5 

2-5 

s-5 
2-5 

2-5 

2-5 
2.O 
2-5 

i-5 
2-5 
2-5 
2-5 

2.0 

2-5 
2  0 
2.0 

i-5 

2.0 
I.O 

2-5 
2-5 
2-5 

13 

3  
4  

14  

15  

16  

1::: 
1  

17  
18 

19  

20  
21   

9  
10 

ii    

(From  Bulletin  No.  65,  Hygienic  Laboratory,  June,  1910,  U.  S.  Public 
Health  and  Marine-Hospital  Service.) 

This,  in  brief,  is  the  theory  and  practice  of  Pasteur's 
system  of  treating  hydrophobia.  It  is  entirely  in  keeping 
with  the  ideas  of  the  present  time.  When  wef  remember 
that  the  first  application  of  the  method  to  human  medicine 
was  made  October  26,  1885,  six  years  before  the  time  we 
began  to  understand  the  production  and  use  of  antitoxins, 


The  Dilution  Method  419 

it  becomes  one  of  the  most  remarkable  achievements  of 
medicine. 

Hogyes  states  that  50,000  persons  in  danger  of  developing 
rabies  have  received  the  treatment  in  the  last  ten  years  and 
that  only  i  per  cent,  have  died. 

The  Dilution  Method. — Hogyes,  of  Budapest,*  believes 
that  Pasteur  was  mistaken  in  supposing  that  the  drying  was 
of  importance  in  attenuating  the  virus,  and  thinks  that 
dilution  is  the  chief  factor.  He  makes  an  emulsion  of  rabbit's 
medulla  (i  gram  of  medulla  to  10  c.c.  of  sterile  broth)  as  a 
stock  solution,  to  be  prepared  freshly  every  day,  and  uses  it 
for  treatment,  the  first  dilution  used  being  i  :  10,000;  then 
on  succeeding  days  i  :  8000,  i  :  6000,  i  :  5000,  i  :  2000, 
i  :  looo,  i  :  500,  i  :  250,  i  :  200,  i  :  100,  and  finally  the  full 
strength,  i  :  10. 

Cabot  t  prepared  a  stock  solution  of  8  parts  of  rabbit's 
brain  and  80  parts  of  glycerin  and  water.  The  quantity 
of  glycerin  added  comprised  one-fifth  of  the  total  bulk. 
After  the  emulsion  was  made  it  was  filtered  through  sterile 
cheese-cloth.  This  emulsion  containing  the  glycerin,  if 
kept  in  the  ice-chest,  will  be  of  standard  virulence  during 
the  entire  period  of-  immunization.  As  the  result  of  his 
experiments,  Cabot  found  the  dilution  method  attended 
with  danger  to  the  animal  immunized,  which  is  not  true  of 
the  dried-cord  method  of  Pasteur.  The  latter  method  is, 
therefore,  the  one  to  be  preferred. 

Though  the  essential  cause  of  rabies  has  not  yet  been  dis- 
covered, its  lesions  can  be  found  in  the  medulla  oblongata 
and  in  the  spinal  ganglia.  These  consist  in  certain  cellular  ag- 
gregations known  as  the  " tubercles  of  Babes, "t  and  in  certain 
degenerative  changes  in  the  ganglionic  nerve-cells.  The  regu- 
larity with  which  these  changes  were  observed  by  Babes  led 
him  to  regard  them  as  useful  for  diagnosticating  the  disease, 
and  Van  Gehuchten  and  Nelis§  and  Ravenel  and  McCarthy 
have  confirmed  this  opinion.  Ravenel  and  McCarthy  ||  think 
that  Babes  gives  undue  prominence  to  the  rabic  tubercle, 
which  consists  of  an  aggregation  of  embryonal  cells  about  the 

*"Acad.  des  Sciences  de  Buda-Pest,"  Oct.  17,  1897;  "Centralbl.  f. 
Bakt.  u.  Parasitenk.,"  1887,  n,  579. 

t  "Journal  of  Experimental  Medicine,"  1899,  vol.  iv,  No.  2. 

t  "Virchow's  Archives,"  ex;  "Ann.  de  1'Inst.,  Pasteur,"  1892,  vi. 

§  "Archiv.  de  Biologic,"  1900,  xvi. 

||  "Trans.  Phila.  Pathological  Society,"  N.  S.,  vol.  m,  1900,  p. 
231;  "University  Medical  Magazine,"  Jan.,  1901. 


42O 


Hydrophobia,  Lyssa,  or  Rabies 


central  canal  of  the  cord,  about  the  ganglionic  nerve-cells, 
and  about  the  capillary  blood-vessels,  but  that  the  lesions  of 
the  nerve-cells  are  pathognomonic  if  taken  in  connection  with 
the  clinical  manifestations  of  the  disease.  The  ganglion-cell 
changes  consist  of  degeneration,  chromatolysis,  and  even 
total  disappearance  of  the  nuclei,  a  dilatation  of  the  peri- 
cellular  space,  and  an  invasion  not  only  of  this  space,  but 
also  of  the  nerve-cells  by  embryonal  cells,  and  at  the  same 
time  the  appearance  of  small  corpuscles  which  are  hyaline, 


$£ 

k  -  '  Vr, **   :-/x  - 


Fig.  131. — Normal  sympathetic 
ganglion  from  a  normal  dog. 
(Modified  from  Crocq.) 


Fig.  132. — Sympathetic  ganglion 
of  a  rabbit  with  experimental  rab- 
ies. (Modified  from  Ravenel  and 
McCarthy.) 


brownish,  and  in  parts  metachromatic.  Spiller*  does  not 
regard  the  lesions  as  pathognomonic  of  rabies. 

If  an  accurate  diagnosis  of  rabies  can  be  made  by  a  sim- 
ple histologic  examination,  in  cases  where  animals  thought 
to  be  mad  have  bitten  human  beings,  much  time  can  be 
saved  in  beginning  the  Pasteur  treatment,  and  probably  a 
number  of  cases  saved. 

Negrif  found  that  by  staining  sections  of  the  cerebrum, 
cerebellum,  pons,  basal  ganglia,  and  sometimes  even  the 

*  "Pathological  Society  of  Philadelphia,"  March,  1901. 
f'Soc.    Med.-Chirurg.    di   Pavia,"    24,   in,    1903;   "Zeitschrift   fiir 
Hygiene,"  etc.,  xun  and  xuv. 


The  Bodies  of  Negri  421 

spinal  ganglia  of  human  beings  or  animals  dead  of  rabies, 
by  Mann's  methylene-blue  and  eosin  method,  it  was  possi- 
ble to  demonstrate  in  the  interior  of  the  nerve-cells  of  their 
protoplasmic  process  red  colored,  rounded  bodies,  measuring 
4  to  10  [i,  as  a  rule,  though  varying  from  i  to  25  /u.  In  ex- 
perimental infections  under  the  dura  they  were  most  num- 


Fig.  133. — Negri  bodies.  Cells  from  Ammon's  horn  impression  of 
cow  133,  dried  and  fixed  with  gentle  heat  and  stained  with  saturated 
alcoholic  eosin  and  Lomer's  alkaline  methylene-blue.  Drawn  with  one- 
twelfth  oil  immersion.  Oc.  3  (Frothingham). 

erous  in  the  hippocampal  convolution.  These  bodies  have 
now  become  known  as  Negri  bodies. 

The  careful  studies  of  Williams  and  Lowden*  convince 
them  that  the  Negri  bodies  are  protozoan  organisms  and 
that  their  presence  is  pathognomonic  of  rabies. 

At  the  State  Live-stock  Sanitary  Board  of  Pennsylvania, 
Reichel  and  Englej  stain  Negri  bodies  with  the  following: 

*  ''Jour.  Infectious  Diseases,"  in,  452,  1906. 
t  Personal  communication. 


422  Hydrophobia,  Lyssa,  or  Rabies 

Sat.  ale.  sol.  methylene  violet 10  c.c. 

Sat.  ale.  sol.  fuchsin 7  drops 

Sterile  water 40  c.c. 

The  smears  of  cerebellum  or  hippocampus  are  fixed  with 
absolute  alcohol  and  ether  and  the  stain  poured  on,  heated, 
poured  back  into  the  bottle,  again  poured  on,  heated  and 
poured  back  into  the  bottle,  this  being  done  three  times,  each 
time  for  about  half  a  minute.  Then  wash  in  water,  blot,  and 
examine.  To  examine,  a  nerve-cell  is  found  with  the  low 
power  and  then  examined  with  the  high  power.  The  Negri 
bodies  are  brick  red.  The  stain  soon  fades.  Smears  kept 
for  any  length  of  time  lose  the  staining  reaction. 

Antirabic  Serum. — Babes  and  Lepp*  thought  that  the 
serum  of  animals  that  have  received  repeated  injections  of 
the  crushed  nervous  tissue  of  rabid  animals  was  neutralizing 
or  destructive  to  the  rabies  virus  in  vitro,  called  it  "anti- 
rabic  serum,"  and  believed  that  it  conferred  a  defensive 
power  upon  other  animals.  Marie  t  found  it  to  be  a  simple 
neurotoxic  serum  and  inert  in  its. action  upon  the  virus. 

*  "Ann.  de  1'Inst.  Pasteur,"  1889,  m. 

t  "  Compt.-rendu  Soc.  Biol.,"  t.  i,vi,  June  18,  1904,  p.  1030. 


CHAPTER   VI. 

CEREBROSPINAL  MENINGITIS. 

DIPLOCOCCUS  INTRACELLULARIS  MENINGITIDIS 
(WEICHSELBAUM)  . 

General  Characteristics. — A  minute  non-motile,  non-flagellate, 
non-sporogenous,  non-chromogenic,  non-liquefying,  aerobic  and  op- 
tionally anaerobic,  pathogenic  coccus,  staining  by  ordinary  methods, 
but  not  by  Gram's  method. 

Acute  cerebrospinal  meningitis  may  be  secondary  to 
various  more  or  less  well-localized  infections  when  it  depends 
upon  such  micro-organisms  as  may  be  carried  by  accident 
to  the  meninges.  Among  these  may  be  mentioned  pneumo- 
cocci,  staphylococci,  streptococci,  Bacillus  influenzae,  B. 
typhosus,  B.  coli,  B.  mallei,  B.  pestis,  and  others. 

In  addition  to  these  cases,  however,  there  are  numerous 
cases  of  primary  infection  of  the  membranes,  either  sporadic 
or  epidemic  in  occurrence.  Such  constitute  the  disease 
known  as  cerebrospinal  fever,  epidemic  cerebrospinal  menin- 
gitis, or  "  spotted  fever."  It  is  a  very  dangerous  febrile 
malady,  characterized  by  high  temperature,  an  irregular 
exanthem,  early  meningitis,  a  moderate  degree  of  contagion, 
and  a  high  mortality.  The  cause  of  this  infection  is  a  specific 
organism  known  as  the  meningococcus,  or  Diplococcus  intra- 
cellularis  meningitidis. 

As  early  as  1887  Weichselbaum*  carefully  described  a 
diplococcus  found  in  6  cases  of  cerebrospinal  meningitis 
that  may  have  been  identical  with  one  found  by  L/eichten- 
stern|  in  1885  in  the  purulent  exudate  of  a  case  of  meningitis, 
and  with  a  coccus  observed  as  early  as  1884  by  Celli  and 
Marchiafava.  J  Weichselbaum 's  studies  and  description  of 
this  coccus  seem  to  have  attracted  but  little  attention  at  first, 
and  references  to  them  are  but  brief  in  most  of  the  text- 
books. The  prevailing  opinion  was  that  its  occurrence  in 

*"Fortschritte  der  Med.,"  x,  18  and  19. 
t  "Deutsche  med.  Wochenschrift,"  1885. 
t  "Gazette  degli  Ospedali,"  1884,  vm. 
423 


424  Cerebrospinal  Meningitis 

cerebrospinal  meningitis  was  accidental,  as  inoculations 
into  animals  showed  its  pathogenic  power  to  be  very  limited. 
The  careful  studies  of  Jager,*  Scherer,f  Councilman,  and 
Mallory  and  Wright  J  (embracing  55  cases,  in  which  the 
cocci  were  found  by  culture  or  by  microscopic  examina- 
tion in  38),  and  of  Flatten, §  Schneider, §  Rieger,§  Schmidt,! 
G6ppert,§  Flugge,§  von  Lingelsheim,  §  and  others.  Bes- 
redka||  and  Flexner**  have,  however,  shown  the  diplococcus 
of  Weichselbaum  to  be,  without  doubt,  the  specific  organism. 


;*e 


* 


Fig.   134. — Meningococcus   in  spinal   fluid    (from    Hiss    and    Zinsser, 
"  Text-Book  of  Bacteriology,"  D.  Appleton  &  Co.,  Publishers). 

Distribution. — The  distribution  of  Diplococcus  intra- 
cellularis  in  nature  is  as  yet  unknown.  It  has  been  found 
in  cerebrospinal  meningitis  by  those  who  have  looked  for 
it,  twice  has  been  found  in  the  nose  in  coryza  by  Scherer, 
has  been  found  in  the  conjunctiva  by  Carl  Frankelf  f  and 
Axenfeld,  J  t  and  in  the  purulent  discharges  of  rhinitis  and 
otitis  by  Jager.  §§ 

Morphology. — The  micro-organism  is  a  biscuit-shaped 
diplococcus  having  a  great  resemblance  to  the  gonococcus. 

*  "Zeitschrift  fur  Hygiene,"  xix,  2,  351. 

t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1895,  xvn,  13  and  14. 

J  "Amer.  Jour.  Med.  Sci.,"  March,  1898,  vol.  cxv,  No.  5. 

§  "Klinisches  Jahrbuch,"  1906. 

||  "Annales  de  1'Inst.  Pasteur,"  1906,  xx,  4. 
**  "Jour.  Kxp.  Med.,"  1906-07. 
ft  "Zeitschrift  fur  Hygiene,"  June  14,  1899. 

\l  Lubarsch  and  Oestertag,  "Ergebnisse  der  allg.  Path.  u.  path. 
Anat.,"  in,  S.  573- 

§§  "Deutsche  med.  Wochenschrift,"  1894,  S.  407. 


Staining  425 

This  resemblance  is  further  increased  by  the  fact  that  the 
cocci  are  usually  found  inclosed  in  the  protoplasm  of  the 
leukocytes.  Weichselbaum,  by  whom  this  was  first  ob- 
served, found  it  constant  in  sections  of  the  brain  and  its 
membranes,  though  in  the  exudate  of  the  disease  a  good 
many  free  cocci  may  be  observed.  It  was  this  peculiar 
relationship  to  the  cells  that  led  Weichselbaum  to  name 
the  organism  Diplococcus  intracellularis.  Many  of  the  cocci 
inclosed  in  the  cells  are  apparently  dead  and  degenerated, 
as  they  stain  badly  and  do  not  grow  when  the  pus  is  trans- 
ferred to  culture-media. 

Identification. — Carl  Frankel,  in  discussing  the  micro- 
organism, points  out  that  its  morphologic  peculiarities  have 
much  in  common  with  the  pneumococcus,  so  that  the  most 
refined  methods  of  differentiation  should  always  precede  a 
positive  determination.  Its  resemblance  to  the  gonococcus 
should  also  be  kept  in  mind. 

Perhaps  the  greatest  difficulty  obtains  in  making  a 
certain  differentiation  between  the  meningococcus  and 
Micrococcus  catarrhalis  (q.  v.),  especially  when  such  investi- 
gations are  intended  as  discovering  the  former  organism  in  the 
nasal  discharges.  This  cannot  be  done  by  microscopic  ex- 
amination, but  must  be  achieved  through  cultivation  of  the 
organisms  and  observation  of  the  cultures.  Micrococcus 
catarrhalis  grows  well  upon  nearly  all  culture-media;  menin- 
gococci,  very  sparsely  except  upon  special  media.  The  former 
organism  grows  fairly  well  at  room  temperatures  (20°  C.  or 
less) ;  the  latter,  only  at  25°  C.  and  above.  The  colonies  of  the 
former  are  coarsely  granular ;  those  of  the  latter,  finely  granu- 
lar. 

Staining. — The  organism  is  easily  stained  with  the  usual 
aqueous  solutions  of  the  anilin  dyes.  According  to  Weich- 
selbaum, Mallory,  and  Wright  it  does  not  stain  by  Gram's 
method. 

For  staining  the  meningococcus  the  method  of  Pick  and 
Jacobsohn*  is  highly  praised  by  Carl  Frankel,  who  modifies 
it  by  adding  three  times  as  much  carbol-fuchsin  as  is  recom- 
mended in  the  original  instructions,  which  are  as  follows: 
Mix  20  c.c.  of  water  with  8  drops  of  saturated  methylene-blue 
solution;  then  add  45  to  50  drops  of  carbol-fuchsin.  Allow 
the  fluid  to  act  upon  the  cover-glass  for  five  minutes.  The 
cocci  alone  are  blue,  all  else  red. 

*  "Berliner  klin.  Wochenschrift,"  1896,  S.  811. 


426  Cerebrospinal  Meningitis 

Isolation. — The  organism  can  be  secured  for  cultivation 
either  from  the  purulent  matter  of  the  exudate  found  at 
autopsy,  or  from  the  fluid  obtained  by  lumbar  puncture.  To 
obtain  this  fluid  Park  *  gives  the  following  directions:  "  The 
patient  should  lie  on  the  right  side  with  the  knees  drawn  up 
and  the  left  shoulder  depressed.  The  skin  of  the  patient's 
back,  the  hands  of  the  operator,  and  the  large  antitoxin 
syringe  should  be  sterile.  The  needle  should  be  4  cm.  in 
length,  with  a  diameter  of  i  mm.  for  children,  and  larger  for 
adults.  The  puncture  is  generally  made  between  the  third 
and  fourth  lumbar  vertebrae.  The  thumb  of  the  left  hand 
is  pressed  between  the  spinous  processes,  and  the  point  of 
the  needle  is  entered  about  i  cm.  to  the  right  of  the  median 
line  and  on  a  level  with  the  thumb-nail,  and  directed  slightly 
upward  and  inward  toward  the  median  line.  At  a  depth  of 
3  or  4  cm.  in  children  and  7  or  8  cm.  in  adults  the  needle 
enters  the  subarachnoid  space,  and  the  fluids  flow  out  in 
drops  or  in  a  stream.  If  the  needle  meets  a  bony  obstruc- 
tion, withdraw  and  thrust  again  rather  than  make  lateral 
movements.  Any  blood  obscures  microscopic  examination. 
The  fluid  is  allowed  to  drop  into  sterile  test-tubes  or  vials 
with  sterile  stoppers.  From  5  to  15  c.c.  should  be  with- 
drawn. No  ill  effects  have  been  observed  from  the  opera- 
tion." 

In  making  a  culture  from  this  fluid  Park  points  out  that, 
as  many  of  its  contained  cocci  are  dead,  a  considerable 
quantity  of  the  fluid  (say  about  i  c.c.)  must  be  used. 

The  cocci  have  also  been  cultivated  from  the  nasal  dis- 
charges in  the  6  cases  studied  by  Weichselbaum  and  in 
1 8  studied  by  Scherer.  Elserf  has  isolated  the  organism 
from  the  circulating  blood  of  patients  suffering  from  epi- 
demic cerebrospinal  fever.  To  determine  the  presence  of 
the  coccus  in  the  nasal  discharges  where  other  similar  cocci 
may  be  present,  Gram's  stain  may  be  used  and  followed  by 
an  aqueous  solution  of  Bismarck-brown.  The  meningococci 
will  be  brown. 

Cultivation. — The  organism  was  successfully  cultivated 
by  Weichselbaum,  but  does  not  readily  adapt  itself  to 
artificial  media.  It  develops  upon  agar-agar  and  glycerin 
agar-agar,  upon  Loffler's  blood-serum  mixture,  and,  accord- 

*  "Bacteriology  in  Medicine  and  Surgery,"  Philadelphia,  1899, 
p.  364. 

t  "Jour.  Medical  Research,"  1906,  xiv,  89. 


Vital  Resistance  427 

ing  to  Goldschmidt,*  upon  potato.  Wiechselbaum  did  not 
find  that  it  developed  upon  potato.  It  does  not  grow  in 
bouillon  or  gelatin.  The  cultures  are  usually  scanty  and 
without  characteristic  features. 

Flexnerf  found  that  the  difficulties  of  cultivation  were 
greatly  reduced  by  the  employment  of  sheep-serum  instead 
of  human  serum.  Sheep-serum  water  was  prepared  accord- 
ing to  the  method  of  Hiss  (sheep-serum  i  part,  water  2 
parts,  sterilized  in  the  autoclave)  and  mixed  with  a  beef- 
infusion  agar-agar  containing  2  per  cent,  of  glucose.  The 
quantity  of  sheep-serum  need  not  exceed  7V  to  •£$  of  the  vol- 
ume of  the  agar-agar.  It  is  added  to  the  sterile  melted 
agar,  which  is  afterward  slanted  in  test-tubes  or  allowed  to 
congeal  on  the  expanded  surface  of  i6-ounce  Blake  bottles 
when  mass  cultures  are  to  be  used.  There  is  nothing  charac- 
teristic about  the  cultures.  The  cocci  grow  only  at  the  tem- 
perature of  the  body,  attain  only  a  sparse  development, 
and  form  a  more  or  less  confluent  line  of  minute,  rounded, 
grayish  colonies  which  are  easily  overlooked  upon  opaque 
media  like  blood-serum.  The  general  characteristics  of  the 
growth  are  not  unlike  those  of  the  pneumococcus,  strep- 
tococcus, and  gonococcus. 

Colonies. — When  grown  upon  agar-agar  plates,  the  deep 
colonies  scarcely  develop  at  all,  appearing  under  the  low- 
power  lens  as  minute,  irregularly  rounded  granular  masses. 
The  surface  colonies  are  larger,  and  consist  of  an  opaque 
yellowish-brown  nucleus  about  which  a  flat,  rounded  disk 
spreads  out.  The  edges  may  be  dentate;  the  color  is  grayish 
or  yellowish  near  the  center,  becoming  less  intense  as  the 
thin  edges  are  reached ;  the  structure  is  finely  granular. 

Vital  Resistance.— The  vitality  of  the  culture  is  low, 
and  the  cocci  die  out  readily,  ceasing  to  grow  when  trans- 
planted after  eight  or  ten  days.  It  becomes  necessary, 
therefore,  when  studying  the  organism  to  transplant  it 
frequently — ParkJ  says  every  two  days.  Flexner§  found 
that  they  do  not  survive  beyond  two  or  three  days  and  that 
transplantations  do  not  succeed  unless  considerable  quanti- 
ties of  the  culture  are  placed  upon  the  surface  of  the  fresh 
medium,  showing  that  many  of  the  organisms  were  already 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  n,  22,  23. 

t  "Jour.  Experimental  Med.,"  1907,  ix,  p.  105. 

|  "Bacteriology  in  Medicine  and  Surgery,"  1899,  p.  362. 

§  Loc.  cit. 


428  Cerebrospinal  Meningitis 

dead.  This  is  confirmed  by  the  microscopic  appearance  of 
the  cultures.  Those  sixteen  to  twenty-four  hours  old  stain 
sharply  and  uniformly;  on  the  second  day  many  of  the  cocci 
show  irregularities  of  size  and  staining,  and  after  several 
days  no  normal-looking  cocci  can  be  found.  It  was  found, 
however,  that  in  carefully  preserved  cultures  of  certain 
strains  a  few  cocci  might  survive  for  many  months.  Vitality 
is  preserved  longest  when  the  cultures  are  kept  in  the 
thermostat  and  not  taken  out  when  grown,  and  kept  at 
room  temperature  or  in  a  refrigerator.  The  addition  of  a 
small  quantity  of  a  calcium  salt  favors  prolonged  vitality 
and  will  sometimes  maintain  it  for  four  or  five  weeks  in  cul- 
tures that  would  otherwise  die  in  a  few  days.  Sodium 
chlorid  is  injurious  to  the  cocci.  Flexner  attributed  the 
autolysis  of  the  cultures  to  an  enzyme. 

The  organism  is  soon  killed  by  drying,  by  exposure  to  the 
sun,  and  by  quite  moderate  variations  of  temperature.  It 
succumbs  to  very  high  dilutions  of  most  germicides  in  a  very 
short  time. 

Agglutination. — When  animals  are  immunized  by  repeated 
injections  of  the  Diplococcus  intracellularis,  their  blood- 
serum  and  body- juices  become  agglutinative.  Such  serums 
kept  in  the  laboratory  can  be  used  for  the  identification  of 
the  coccus  in  fresh  culture,  though  the  reaction  is  not  exact, 
since  the  agglutinability  of  different  strains  of  cocci  is  differ- 
ent. The  serums  have  an  agglutinating  power  that  varies 
from  i :  500  to  i :  3000  in  the  hands  of  different  observers. 

Metabolic  Products. — The  meningococcus  produces  an 
endotoxin.  Albrech  and  Ghon*  were  able  to  kill  white  mice 
with  dead  cultures.  Lepierre  |  obtained  a  toxin  from  bouillon 
cultures  by  precipitating  them  with  alcohol. 

Pathogenesis. — The  results  of  animal  inoculations  made 
with  Diplococcus  intracellularis  meningitidis  are  disappoint- 
ing. Subcutaneous  inoculations  into  the  lower  animals  are 
continually  without  effect.  Intrapleural  and  intraperito- 
neal  injections  of  cultures  of  the  organism  into  mice  and 
guinea-pigs  are  sometimes  fatal,  the  dead  animals  showing 
a  serofibrinous  inflammation  with  the  presence  of  the  cocci. 
The  intravenous  injection  of  the  coccus  into  rabbits  is 
followed  by  death  without  important  or  conclusive  symp- 
toms, and  usually  without  the  presence  of  cocci  in  the  blood. 

*  "Wiener  klin.  Wochenschrift,"  1901. 

t  "  Jour,  de  phys.  et  de  path,  gen.,"  v,  No.  3. 


Pathogenesis  429 

Weichselbaum  endeavored  to  reproduce  the  original 
cerebrospinal  meningitis  in  animals  by  trephining  and  in- 
jecting the  cocci  beneath  the  dura.  In  this  manner  he 
inoculated  three  rabbits  and  three  dogs.  Two  of  the  rabbit 
injections  failed,  probably  because  the  injected  material 
escaped  at  once  from  the  wound.  The  third  rabbit  died, 
and  showed  marked  congestion  of  the  membranes  of  the 
brain  and  a  minute  softened  and  hemorrhagic  area.  In 
these  the  cocci  were  found  by  culture  to  be  abundant. 
The  three  dogs  all  died  with  congestion  and  pus-formation 
in  the  membranes  and  areas  of  softening  in  the  brain  sub- 
stance. The  cocci  were  recovered  from  two  of  the  dogs, 
but  the  lesions  of  the  third  animal,  which  lived  twelve  days, 
contained  none. 

Flexner*  found  that  in  large  doses  the  coccus  was  always 
capable  of  killing  small  guinea-pigs  and  mice  when  injected 
intraperitoneally.  To  achieve  this,  however,  the  organisms 
should  be  suspended  in  sheep-serum  water,  not  in  salt 
solution,  which  is  an  active  poison  to  the  coccus. 

Bettencourt  and  Franca f  tried  to  infect  monkeys  by 
trephining,  by  injecting  into  the  spinal  canal,  and  by  rubbing 
the  cocci  upon  the  nasal  mucous  membranes,  but  without 
success.  Von  Lingelsheim  and  Leuchs|  and  Flexner  §  were 
more  successful.  Flexner 's  method  was  to  introduce  a  hypo- 
dermic needle  into  the  spinal  canal,  wait  until  a  few  drops  of 
cerebrospinal  fluid  had  escaped,  and  then  inject  the  culture. 
When  thus  introduced  at  a  low  level  of  the  spinal  canal,  the 
diplococci  distribute  themselves  through  the  meninges  in  a 
few  hours  and  excite  an  acute  meningitis,  the  exudate  of 
which  accumulates  chiefly  in  the  lower  spinal  meninges  and 
the  meninges  of  the  base  of  the  brain.  The  inflammation 
extends,  in  monkeys,  into  the  membranes  covering  the 
olfactory  lobes  and  along  the  dura  mater  into  the  ethmoid 
plate  and  nasal  mucosa. 

The  nasal  mucous  membrane  is  found  in  many  instances 
to  be  inflamed  and  beset  with  hemorrhages.  Smear  prepara- 
tions from  the  nasal  mucosa  show  many  polymorphonuclear 
leukocytes  containing  the  cocci  in  a  degenerated  form.  The 
cocci  were  not  cultivated  from  the  nasal  exudates. 

*  Loc  cit. 

t  "Zeitschr.  f.  Hyg.  u.  Infekt.,"  XL,VI,  p.  463. 

J  "Klin.  Jahrbuch,"  1906,  xv,  p.  489. 

§  Loc.  cit. 


430  Cerebrospinal  Meningitis 

Mode  of  Infection. — It  is  not  known  by  what  channels 
infection  with  Diplococcus  intracellularis  meningitidis  takes 
place.  Weichselbaum  supposed  it  might  enter  by  the  nasal, 
auditory,  or  other  passages,  especially  the  nose,  where  he 
constantly  found  it,  and  the  more  recent  studies  of  Goodwin 
and  Sholly*  have  shown  the  organisms  to  be  of  frequent 
occurrence  in  the  nasal  cavities  of  meningitis  patients  as 
well  as  occasionally  in  those  associated  with  them.  It  thus 
becomes  evident  that  association  with  the  diseased  may 
lead  to  the  infection  of  the  well,  and  that  the  cases  should 
be  isolated.  The  same  conclusions  were  reached  by  Kolle 
and  Wassermann,f  who  studied  the  nasal  secretions  of  112 
healthy  individuals,  not  exposed  to  the  disease,  without 
finding  any  cocci,  but  found  them  in  the  nasopharynx  of 
the  father  of  a  child  suffering  from  the  disease,  and  that  of 
another  child  with  suspicious  symptoms. 

Steel  J  has  found  what  may  be  a  variety  of  the  meningo- 
coccus  in  the  simple  posterior  basic  meningitis  of  infants. 
The  organism  differs  from  that  of  Weichselbaum  in  having 
a  greater  longevity  upon  culture-media,  where  it  often  lives 
as  long  as  thirty  days.  It  is  easily  stained  by  methylene- 
blue,  but  not  by  Gram's  method.  Another  similar  organism 
has  been  described  by  Elser  and  Huntoon.§ 

Specific  Therapy. — Kolle  and  Wassermann||  carefully 
studied  antimeningococcus  sera  for  specific  opsonins,  for 
bacteriotropic  substances,  and  for  other  evidences  of  favor- 
able therapeutic  action,  but  came  to  no  definite  conclusions. 
Flexner  and  Jobling  **  had  better  success  both  in  developing 
the  experimental  and  practical  knowledge  of  the  serum. 
The  serum  was  prepared  first  with  goats  and  then  with 
horses,  the  animals  being  injected  with  suspensions  of  the 
meningococci.  The  serum  is  used  by  injecting  it  into  the 
spinal  canal  through  a  lumbar  puncture.  The  precaution 
must  be  taken  to  permit  some  of  the  fluid  to  escape  first,  and 
then  replace  it  by  the  antiserum,  of  which  not  more  than 
30  c.c.  must  be  injected.  Tabulations  of  the  results  follow- 
ing the  employment  of  Flexner 's  serum  show  a  large  per- 
centage of  recoveries. 

*  ''Journal  of  Infectious  Diseases,"  1906,  Supplement  No.  2,  p.  21. 
f  "Klinisches  Jahrbuch,"  xv,  1906. 
t  " Pediatrics, "  Nov.  15,  1898. 
§  "Journal  of  Medical  Research,"  1909,  xx,  377. 
||  Loc.  cit. 
**  "Jour.  Experimental  Medicine,"  1907,  ix,  p.  168,  and  1908,  x,  p.  141. 


CHAPTER   VII. 
GONORRHEA. 

MICROCOCCUS  GONORRHOEA  (NEISSER). 

,  General  Characteristics.— A  minute,  biscuit-shaped,  non-motile, 
non-sporogenous,  non-liquefying,  non-chromogenic,  non-flagellate,  aero- 
bic, strictly  parasitic  coccus,  not  stained  by  Gram's  method,  cul- 
tivable upon  special  media,  and  pathogenic  for  man  only. 

All  authorities  now  accept  the  "  gonococcus "  as  the 
specific  cause  of  gonorrhea.  It  was  first  observed  in  the 
urethral  and  conjunctival  secretions  of  gonorrhea  and 
purulent  ophthalmia  by  Neisser*  in  1879. 

Bumm|  found  other  cocci  closely  resembling  the  gono- 
coccus in  the  inflamed  urethra,  and  points  out  that  neither 
its  shape  nor  its  position  in  the  cells  can  be  regarded  as 
characteristic,  but  that  failure  to  stain  by  Gram's  method 
can  alone  enable  us  to  say  with  certainty  that  biscuit-shaped 
cocci  found  in  urethral  pus  are  gonococci. 

Distribution. — The  gonococcus  is  a  purely  parasitic 
pathogenic  organism.  It  can  be  found  in  the  urethral  dis- 
charges of  gonorrhea  from  the  beginning  until  the  end  of  the 
disease,  and  often  for  many  months  and  even  years  after  re- 
covery from  it.  After  the  period  of  creamy  pus  has  passed,  its 
numbers  are  usually  outweighed  by  other  pyogenic  organisms. 
WertheimJ  cultivated  the  gonococcus  from  a  case  of  chronic 
urethritis  of  two  years'  standing,  and  proved  its  virulence 
by  producing  experimental  gonorrhea  in  a  human  being. 
The  organisms  are  chiefly  found  within  the  pus-cells  or 
attached  to  the  surface  of  epithelial  cells,  and  should 
always  be  sought  for  as  diagnostic  of  gonorrhea,  as  purulent 
urethritis  is  sometimes  caused  by  other  organisms,  as 
Bacillus  ooli  communis§  and  Staphylococcus  pyogenes. 

*  "Centralbl.  f.  d.  med.  Wissenschaft,"  1879,  No.  28. 

t"Der  Mikroorganismus  der  gonorrhoischen  Schleimhauterkrank- 
ungen,"  "Gonococcus  Neisser,"  second  edition,  1887. 

t  "Archiv  f.  Gynakologie,"  Bd.  xui,  1892,  Heft  i. 

§  Van  der  Pluyn  and  Loag,  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Bd. 
xvn,  Nos.  7,  8,  Feb.  28,  1895,  p.  233. 


432  Gonorrhea 

Morphology. — The  organisms  occur  in  pairs.  Bach  pair 
of  young  cocci  is  composed  of  two  spherical  organisms,  but 
as  they  grow  older  the  inner  surfaces  become  flattened  and 
separated  from  one  another  by  a  narrow  interval,  so  that 
they  somewhat  resemble  a  coffee-bean.  Sometimes  tetrads 
are  seen,  the  group  no  doubt  resulting  from  the  division  of 
a  pair.  A  pair  of  the  cocci  resembles  the  German  biscuit, 
and  is  described  by  the  Germans  as  semmelfdrmig. 

The  gonococci  are  small,  the  length  of  one  of  the  coffee- 
bean  cocci  being  about  1.6  ^,  its  breadth  about  0.8  ^.  They 
are  not  motile,  nor  provided  with  flagella,  and  are  without 
spores. 

Quite  as  characteristic  as  the  form  of  the  organism  is  its 
relation  to  the  cells.  In  most  of  the  inflammatory  exudates 


Fig-  135- — Gonococci  in  urethral  pus. 

the  gonococci  are  contained  either  in  epithelial  cells  or  in 
leukocytes,  very  few  of  them  lying  free.  This  intracellular 
position  is  supposed  to  depend  upon  active  phagocytosis  of 
the  cocci  by  the  cells.  It  may  not  obtain  in  old  lesions. 

Staining. — They  stain  readily  with  all  the  aqueous  solu- 
tions of  the  anilin  dyes — best  with  rather  weak  solutions, 
but  not  by  Gram's  method. 

The  organisms  contained  in  pus  can  be  beautifully  shown 
by  first  treating  the  prepared  film  with  alcoholic  eosin,  and 
then  with  LofHer's  alkaline  methylene-blue.  A  differential 
color  test  can  be  made  by  staining  the  film  by  Gram's  method 
and  then  with  aqueous  Bismarck-brown,  or,  what  may  be  still 
better,  with  3  per  cent,  aqueous  solution  of  pyronin.  Ordi- 


Isolation  and  Cultivation  433 

nary  pus  cocci,  taking  the  Gram's  stain,  appear  blue-black; 
the  gonococci  are  dark  brown. 

Isolation  and  Cultivation. — The  cultivation  of  the  gono- 
coccus  is  difficult  and  requires  considerable  bacteriologic 
skill. 

The  organism  does  not  grow  upon  any  of  the  ordinary 
culture-media,  and  grows  very  scantily  upon  any  artificial 
medium.  Wertheim*  succeeded  in  cultivating  it  by  diluting 
a  drop  of  gonorrheal  pus  with  human  blood-serum,  mixing 
this  with  an  equal  part  of  melted  2  per  cent,  agar-agar  at 
40°  C.,  and  pouring  the  mixture  into  Petri  dishes,  which, 
as  soon  as  the  medium  became  firm,  were  stood  in  the 
incubator  at  37°  C.  or,  preferably,  40°  C.  In  twenty-four 
hours  the  colonies  could  be  observed.  Those  upon  the  sur- 
face showed  a  dark  center,  surrounded  by  a  delicate  granu- 
lar zone. 

Young  f  had  excellent  success  with  a  hydrocele-agar  pre- 
pared as  follows:  "  The  fluid  (hydrocele  or  ascitic)  is  ob- 
tained sterile,  the  locality  of  the  puncture  being  carefully 
sterilized  by  modern  surgical  methods,  the  sterile  trocar 
covered  at  its  external  end  with  sterilized  gauze  so  as  not  to 
be  infected  by  the  operator's  hand,  and  the  fluid  collected  in 
sterile  flasks,  the  sterile  stoppers  being  then  replaced.  Col- 
lecting the  fluid  in  .this  way  we  have  very  rarely  had  it  con- 
taminated, often  keeping  it  several  months  before  using  it. 
The  fluid  is  mixed  with  ordinary  nutrient  agar.  A  number 
of  common  slants  are  put  in  the  autoclave  for  five  minutes. 
This  liquefies  the  agar  and  at  the  same  time  thoroughly 
sterilizes  the  tubes  and  cotton  stoppers.  The  slants  are 
then  put  in  a  water-bath  at  55°  C.  so  as  not  to  coagulate  the 
albumin  when  mixed  with  the  agar.  The  stopper  having 
been  removed  from  a  small  flask  of  hydrocele  fluid,  the  top 
of  the  flask  is  flamed  and  the  albuminous  fluid  is  then  poured 
into  an  agar  tube  (the  top  of  which  has  also  been  flamed)  in 
proportions  a  little  more  than  one  to  two."  The  medium 
can  be  allowed  to  solidify  in  tubes  or  can  be  poured  into 
Petri  dishes. 

When  one  of  the  colonies  was  transferred  to  a  tube  of 
human  blood-serum,  or  of  one  of  the  above-described  mix- 
tures obliquely  coagulated,  isolated  little  gray  colonies  occur, 

*  "Archiv.  fur  Gynakologie,"  1892. 

f  "Contributions  to  the  Science  of  Medicine  by  the  Pupils  of  Wil- 
liam M.  Welch,"  Baltimore,  1900,  p.  677. 

28 


434  Gonorrhea 

later  becoming  confluent  and  producing  a  delicate  smeary 
layer  upon  the  medium.  The  main  growth  is  surrounded  by 
a  thin,  veil-like  extension  which  gradually  fades  away  at 
the  edges.  A  slight  growth  also  occurs  in  the  water  of 
condensation. 

Heiman*  found  that  the  gonococcus  grows  best  in  a 
mixture  of  i  part  of  pleuritic  fluid  and  2  parts  of  2  per 
cent.  agar.  Wright  t  prefers  a  mixture  of  urine,  blood- 
serum,  peptone,  and  agar-agar. 

Wassermann  t  used  a  mixture  of  15  c.c.  of  pig-serum, 
35  c.c.  of  water,  3  c.c.  of  glycerin,  and  2  per  cent,  of  nutrose. 
The  nutrose  is  dissolved  by  boiling  and  the  solution  sterilized. 
This  is  then  added  to  agar,  in  equal  parts,  and  used  in  plates.  § 

Laitinen||  found  agar-agar  mixed  with  one-third  to  one- 
half  its  volume  of  cyst  or  ascitic  fluid,  and  bouillon  con- 
taining i  per  cent,  of  peptone  and  0.5  per  cent,  of  sodium 
chlorid,  mixed  with  one-third  to  one-half  its  volume  of 
cyst  or  ascitic  fluid,  very  satisfactory.  The  gonococcus 
could  be  kept  alive  upon  these  media  for  two  months., 
Laitinen  found  that  the  gonococcus  produces  acids  in  the 
early  days  of  its  development,  and  alkalies  subsequently. 
He  was  unable  to  isolate  any  toxin  from  the  cultures. 

Vital  Resistance. — Authorities  agree  that  the  gonococ- 
cus has  very  slight  power  of  heat  endurance.  Wertheim 
found  the  optimum  temperature  of  cultivation  to  be  39°  to 
40°  C.,  and  saw  no  harm  result  from  exposure  to  42°  C.  It 
is,  however,  doubtful  whether  this  temperature  can  be  long 
survived  and  whether  higher  temperatures  can  be  endured. 
The  gonococci,  though  not  easily  cultivated,  are  said  to 
resist  unfavorable  conditions,  especially  drying,  very  well. 
Kratter  was  able  to  demonstrate  their  presence  upon  washed 
clothing  after  six  months,  and  found  that  they  still  stained 
well.  This  may  not  mean  that  the  organisms  were  still 
alive. 

In  artificial  culture  the  gonococcus  soon  dies,  though 
cultures  from  different  sources  differ  considerably  in  this 
regard.  As  a  rule  they  survive  but  a  few  transplantations, 

*  "Medical  Record,"  Dec.  19,  1886. 
f  "Amer.  Jour.  Med.  Sci.,"  Feb.,  1895. 
t  "Berliner  klin.  Wochenschrift,"  1897. 

§  See  "  Text-Book  of  Bacteriology,"  by  Hiss  and  Zinsser,  1910,  p.  383, 
||  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  June  i,  1898,  vol.  xn,  No.  20, 
p.  874- 


Toxic  Products  435 

though  Young  found  that  one  culture  had  been  kept  alive 
by  students  in  his  laboratory  for  more  than  three  months. 

Diagnosis. — The  diagnosis  of  gonorrhea  by  finding  the 
diplococci  in  the  pus  and  epithelial  cells  is  a  very  simple 
matter.  The  certain  recognition  of  the  micro-organisms 
under  other  conditions  is  by  no  means  an  easy  matter. 
Thus,  when  gonorrhea  becomes  chronic  and  the  cocci  are 
no  longer  taken  up  by  the  phagocytes,  and  so  lose  their 
intracellular  occurrence,  it  raises  a  little  doubt  whether 
Gram-negative  cocci  may  be  true  gonococci  or  not,  yet  it  is 
at  precisely  this  time  when  a  patient  getting  over  gleet  and 
wanting  to  marry  desires  to  know  definitely  whether  gono- 
cocci are  any  longer  present  in  his  urethra  or  not.  Again, 
when  the  gonococcus-like  organisms  occur  upon  the  conjunc- 
tiva, in  the  pus  taken  from  joints,  from  the  valves  of  the 
heart,  or  from  the  Fallopian  tubes,  the  same  difficulty  is  met. 
Probably  the  greatest  perplexity  arises  when  the  conjunctiva 
is  called  in  question,  for  here  there  can  come  about  a  confu- 
sion of  the  gonococcus,  the  pneumococcus,  and  Micrococcus 
catarrhalis  (q.  v.)  which  only  careful  staining  and  culture 
experiments  can  solve.  The  pneumococcus  may  be  readily 
separated  if  its  lanceolate  form  and  capsules  can  be  observed, 
but  it  is  only  by  seeing  that  Micrococcus  catarrhalis  grows 
readily  and  luxuriantly  upon  all  the  laboratory  media, 
and  the  gonococcus  with  difficulty  and  very  sparingly  upon 
any  media,  that  the  diagnosis  can  be  made  with  certainty. 

The  method  of  diagnosis  by  staining  and  looking  for 
Gram-negative  diplococci  in  the  cells  is  only  a  "  rough  and 
ready  "  one  and  is  not  dependable. 

Toxic  Products. — The  toxic  metabolic  products  of  the 
gonococcus  appear  to  be  contained  within  the  bodies  of  the 
bacteria  and  disseminated  but  slightly  throughout  the 
culture-media.  Christmas,*  Nicolaysen,f  and  Wasser- 
mannj  have  studied  gonotoxin,  and  have  all  found  that  it 
remains  in  the  bodies  of  the  bacteria.  The  toxin  seems 
to  be  quite  stable  and  is  not  destroyed  by  temperatures 
fatal  to  the  cocci.  Wassermann  obtained  some  cultures  of 
which  o.i  c.c.  would  kill  mice;  others,  of  which  i.o  c.c. 

*  ''Ann.  de  1'Inst.  Pasteur,"  1897. 

t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1897,  Bd.  xxn,  Nos.  12 
and  13,  p.  305. 

I  "Zeitschrift  fiir  Hygiene,"  1898,  and  "Berliner  klin.  Wochen- 
schrift,"  1897,  No.  32,  p.  685. 


436  Gonorrhea 

was  required.  The  poison  can  be  precipitated  with  absolute 
alcohol.  Small  quantities  of  the  toxin  introduced  into  the 
urethra  cause  suppuration  at  the  point  of  application,  fever, 
swelling  of  the  neighboring  lymphatic  nodes,  and  muscular 
and  articular  pains, 

Pathogenesis. — It  is  generally  believed  that  gonorrhea 
cannot  be  communicated  to  animals. 

There  is  no  doubt  but  that  the  gonococcus  causes  gonor- 
rhea, as  it  has  on  several  occasions  been  intentionally  and 
experimentally  inoculated  into  the  human  urethra  with 
resulting  typical  disease.  It  is  constantly  present  in  the 
disease,  and  very  frequently  in  its  sequelae,  though  it  not 
infrequently  happens  that  the  lesions  secondary  to  gonor- 
rhea are  caused  by  the  more  common  organisms  of  suppu- 
ration that  have  entered  through  the  surface  denudations 
caused  by  the  gonococcus. 

The  mucous  membranes,  especially  those  covered  with 
squamous  epithelium,  are  the  appropriate  portals  for  gonor- 
rheal  infection. 

The  injection  of  gonococci  into  the  subcutaneous  tissue  is 
not  followed  by  either  abscess  formation  or  septic  infection. 

Gonococci  may  enter  the  circulation  of  human  beings  and 
occasion  a  peculiar  septic  condition  with  irregular  tempera- 
ture, apt  to  be  followed  by  invasion  of  the  cardiac  valves, 
joints,  or  other  tissues.  P.  Kraus*  has  twice  succeeded  in 
cultivating  the  gonococcus  from  the  blood  of  patients  in  the 
stage  of  septic  infection. 

The  deep  lesions  caused  by  the  gonococcus  are,  however, 
numerous,  and  in  Young's  paper  (loc.  cit.)  its  widespread 
powers  of  pyogenic  infection  are  well  shown  in  a  collection 
of  the  cases  recorded  in  the  literature,  and  some  original 
observations  showing  the  undoubted  occurrence  of  the  gono- 
coccus in  gonorrhea,  ophthalmia  neonatorum,  arthritis,  ten- 
dosynovitis,  perichondritis,  subcutaneous  abscess,  intra- 
muscular abscess,  salpingitis,  pelvic  peritonitis,  adenitis, 
pleuritis,  endocarditis,  septicemia,  acute  cystitis,  chronic 
cystitis,  pyonephrosis,  and  diffuse  peritonitis. 

In  the  beginning  of  the  inflammatory  process  the  cocci 
grow  in  the  superficial  epithelial  cells,  but  soon  penetrate 
between  the  cells  to  the  deeper  layers,  where  they  continue 
to  keep  up  the  irritation  as  the  superficial  cells  desquamate. 
Opinions  differ  as  to  whether  the  gonococci  can,  with  equal 
*  "Berliner  klin.  Wochenschrift,"  No.  19,  p.  494,  May  9,  1904. 


Immunization  437 

facility,  penetrate  squamous  and  columnar  epithelium. 
Their  attacks  are  usually  made  upon  surfaces  covered  with 
squamous  epithelium. 

All  urethral  inflammations,  and  in  gonorrhea  all  of  the 
inflammatory  symptoms,  do  not  depend  upon  the  gonococcus. 
The  periurethral  abscess,  salpingitis,  etc.,  not  infrequently 
depend  upon  ordinary  pus  cocci,  and  I  remember  having 
seen  a  case  of  gonorrhea  with  double  orchitis,  general  septic 
infection,  and  endocarditis  in  which  the  gonococci  had  no 
role  in  the  sepsis,  which  was  caused  by  a  large  dumb-bell 
coccus  that  stained  beautifully  by  Gram's  method. 

In  the  remote  secondary  inflammations  the  gonococci 
disappear  after  a  time,  and  the  inflammation  either  subsides 
or  is  maintained  by  other  bacteria.  In  synovitis,  however, 
the  inflammation  excited  may  last  for  months. 

So  long  as  the  gonococci  persist  in  his  urethra  or  other 
superficial  tissues  the  patient  may  spread  the  contagion, 
and  after  apparent  recovery  from  gonorrhea  the  cocci  may 
remain  latent  in  the  urethra  for  years,  not  infrequently 
causing  a  relapse  if  the  patient  partake  of  some  substance, 
as  alcohol,  irritating  to  the  mucous  membranes.  Bearing 
this  in  mind,  physicians  should  be  careful  that  their  patients 
are  not  too  soon  discharged  as  cured  and  permitted  to 
marry. 

Immunization  against  the  gonococcus  has  not  yet  been 
successfully  achieved.  Wassermann  failed  altogether ;  Christ- 
mas claims  to  have  immunized  goats,  but  the  serum  of  these 
animals  could  not  be  shown  to  contain  any  antitoxin  or  to 
be  bacteriolytic. 

Torrey*  prepared  an  antigonococcus  serum  by  immunizing 
rabbits  with  gonotoxin.  The  culture  used  was  isolated  from 
a  case  of  acute  gonorrhea  in  a  medium  of  rich  ascitic  fluid 
and  slightly  acid  beef  infusion,  peptone  broth,  equal  parts. 
In  speaking  about  this  mixture  Dr.  Torrey  told  me  that 
the  exact  reaction  was  its  most  important  feature,  as  other- 
wise the  gonococci  soon  died.  Tubes  of  about  12  cm.  of 
the  mixture  were  heated  to  about  60°  C.  for  several  hours 
and  then  tested  for  sterility.  The  cocci  were  cultivated  at 
36°  to  37°  C.  After  eighteen  to  twenty-four  hours'  incuba- 
tion a  slight  granular  growth  appears  near  the  surface  and  on 
the  sides  of  the  tube.  This  slowly  increases  until  after  six 
days  the  medium  is  well  clouded  on  shaking.  Large  rab- 
*  "Journal  Amer.  Med.  Assoc.,"  Jan.  27,  1906,  XL,VI,  p.  261. 


438  Gonorrhea 

bits  were  used  for  making  the  serum,  and  were  intraperi- 
toneally  inoculated  with  10  c.c.  of  an  entire  culture.  The 
first  inoculation  resulted  in  a  loss  of  weight,  sometimes 
amounting  to  one-fourth  of  the  body-weight.  After  an 
interval  of  five  or  six  days  a  second  injection  is  given,  then 
after  a  similar  interval,  a  third,  and  so  on.  The  best  results 
were  obtained  when  cultures  from  six  to  fifteen  days  old 
were  employed.  The  rabbits  were  bled  for  the  first  time 
after  the  sixth  dose,  as  if  the  treatment  be  pushed  they 
soon  fall  into  a  state  of  cachexia,  rapidly  emaciate,  and  die. 
Each  animal  furnishes  70  to  90  cm.  of  the  serum,  which  was 
inclosed  in  2 -cm.  bulbs,  hermetically  sealed,  and  kept  without 
any  preservative. 

With  serum  made  in  this  way  by  Torrey,  Rogers*  treated 
a  number  of  obstinate  cases  of  gonorrheal  rheumatism,  with 
most  satisfactory  results. 

Good  results  in  gonorrheal  arthritis  and  in  gleet  are  also 
claimed  for  treatment  with  gonococco-vaccines  prepared  as 
suggested  by  A.  E.  Wright. 

*  "Jour.  Amer.  Med.  Assoc.,"  XLVI,  p.  261,  Jan.  27,  1906. 


CHAPTER  VIII. 

CATARRHAL  INFLAMMATION. 

MICROCOCCUS  CATARRHALIS  (SEIFERT). 

General  Characteristics.— A  small,  slightly  ovoid,  non-motile, 
non-sporulating,  non-flagellated,  non-liquefying  aerobic  and  optionally 
anaerobic,  non-chromogenic  coccus,  pathogenic  for  man,  and  not  for  the 
lower  animals,  cultivable  upon  the  ordinary  media,  staining  by  the 
ordinary  methods,  but  not  by  Gram's  method. 

This  micro-organism,  which  seems  to  be  closely  related  to 
the  staphylococci,  was  first  observed,  in  sections  of  the  lung 
of  a  case  of  influenza,  by  Seifert.*  It  was  successfully  culti- 
vated in  1890  by  Kirchnerf  from  10  cases  of  an  influenza- 
like  affection.  It  has  since  been  frequently  demonstrated 
in  the  exudates  from  various  inflammatory  conditions  of  the 
respiratory  tract  and  conjunctiva,  and  seems  to  be  a  not 
uncommon  organism  of  superficial  inflammations.  It  is 
a  rather  troublesome  organism,  causing  some  confusion 
because  of  its  disposition  to  occur  in  pairs,  which  gives  it  a 
close  resemblance  to  the  pneumococcus  except  in  cases  in 
which  the  capsules  of  the  latter  are  distinct.  It  is  also 
readily  taken  up  by  the  leukocytes,  and  may  so  resemble 
the  gonococcus;  and  it  is  not  always  easy,  perhaps  not 
always  possible,  to  distinguish  it  from  the  Diplococcus 
intracellularis  meningitidis. 

Morphology. — The  organism  is  spheric  or  slightly  ovoid, 
may  occur  singly,  though  usually  appears  in  pairs  or  clus- 
ters. Large  numbers  are  enclosed  in  the  leukocytes  or  other 
cells.  The  spheric  organisms  have  a  diameter  of  about 

1  ^;  the  ovoid  organisms  may  measure  as  much  as  1.5  by 

2  li.     The  relation  of  the  cocci  to  the  cells  seems  to  have 
something  to  do  with  the  course  of  the  inflammatiory  con- 
ditions with  which  they  are  associated.     During  the  activity 
of  the  process  large  numbers  of  the  cocci  may  be  free ;  toward 
its  close  they  may  all  be  enclosed  in  the  leukocytes. 

*  "Volkmann's  klin.  Vortr.,"  Nr.  240. 
t  "Zeitschr.  f.  Hyg.,"  Bd.  9. 
439 


44°  Catarrhal  Inflammation 

The  organisms  are  not  motile  and  they  have  no  flagella. 

Staining. — The  cocci  stain  by  ordinary  methods,  but  not 
by  Gram's  method. 

Cultivation. — The  organism  can  be  easily  cultivated, 
and  thus  differentiates  itself  from  the  fastidious  gonococcus. 
The  colonies  are  large,  white,  irregular  in  outline,  elevated  at 
the  center,  not  viscid,  and  grow  readily  at  room  temperatures 
upon  all  the  culture-media,  the  best  upon  blood  agar-agar. 
The  vitality  of  the  organism  in  culture  is  not  great.  Very 
often  transplantations  made  after  from  four  to  six  days  fail 


Fig.  136. — Micrococcus  catarrhalis  in  smear  from  sputum  (F.  T.  Lord; 
photo  by  L.  S.  Brown). 

to  grow;  and  in  the  cultures  one  usually  finds  many  deeply 
staining,  supposedly  living  cocci,  and  many  poorly  staining, 
supposedly  dead  organisms. 

Agar=agar. — The  culture  in  general  resembles  that  of 
Staphylococcus  albus.  When  blood  is  added  to  the  agar- 
agar,  the  growth  is  more  luxuriant,  whitish,  and  usually 
consists  of  closely  approximated  colonies  which  do  not 
become  confluent. 

Gelatin. — This  medium  is  not  liquefied. 

Bouillon. — At  the  end  of  the  first  day  no  growth  seems 


Pathogenesis 


441 


to  have  taken  place,  but  at  the  end  of  the  second  day  there 
is  a  slight  clouding  and  a  meager  precipitate.  The  organism 
seems  to  maintain  its  vitality  somewhat  longer  in  bouillon 
than  in  other  culture-media. 

Pathogenesis. — The  organism  seems  to  be  scarcely  patho- 
genic for  animals.  Kirchner  was  able  to  kill  a  guinea- 
pig  by  intrapleural  injection,  and  Neisser,  who  performed 
numerous  experiments  upon  mice,  guinea-pigs,  and  rabbits, 


Fig.  137. — Micrococcus  catarrhalis  colonies  on  agar  (F.  T.  Lord;  photo 
by  L.  S.  Brown). 


only  once  succeeded  in  producing  a  fatal  infection,  by  the 
intraperitoneal  injection  of  0.4  c.c.  of  bouillon  culture.  In 
this  animal  the  cocci  were  found  in  all  the  internal  organs. 
As  has  already  been  said,  the  organism  is  found  associated 
with  superficial  inflammatory  conditions  of  the  mucous 
membrane.  It  is  probably  most  common  in  influenza.  It 
has  also  been  found  in  conjunctivitis,  in  bronchitis,  in  whoop- 
ing-cough, and  in  pneumonia. 


CHAPTER   IX. 
CHANCROID. 

THE  BACILLUS  DUCREYI. 

General  Characteristics. — A  small,  ovoid  streptobacillus,  with 
rounded,  deeply  staining  ends,  non-motile,  non-flagellate,  non-sporog- 
enous;  aerobic  and  optionally  anaerobic,  non-chromogenic,  staining 
by  ordinary  methods,  but  not  by  Gram's  method,  cultivable  on  special 
media  only  and  pathogenic  only  for  man  and  certain  monkeys. 

The  chancroid,  soft  chancre,  or  non-specific  sore,  as  it  is 
called,  is  a  common  venereal  affection  of  both  sexes,  most 
frequent  among  those  who  give  little  attention  to  cleanli- 
ness. It  is  characterized  by  the  appearance  of  a  soft  reddish 
papule,  which  makes  its  appearance  usually  upon  the  genital 
organs,  rarely  upon  other  parts  of  the  body,  soon  after  the 
infection,  and  soon  becomes  transformed  to  an  ugly  ulcera- 
tion  whose  usual  tendency  is  toward  slow  and  persistent 
enlargement,  though  in  different  cases  it  may  be  indolent, 
active,  phagedenic,  or  serpiginous.  The  inguinal  or  other 
nearby  lymph-nodes  early  enlarge  and  soon  soften  and 
ulcerate.  The  disease  is,  therefore,  extremely  destructive 
to  the  tissues  invaded,  though  no  constitutional  involvement 
ever  takes  place. 

Specific  Organism. — In  1889  Ducrey*  described  a  peculiar 
organism  whose  presence  he  was  able  to  demonstrate  with 
great  constancy,  sometimes  in  pure  culture,  in  the  lesions 
of  chancroid,  and  which  he  believed  to  be  the  specific  or- 
ganism of  the  affection.  Unnaf  later  described  an  organ- 
ism resembling  that  of  Ducrey,  and  the  later  observa- 
tions of  Krefting,  {  Peterson, §  Nicolle,||  Cheinisse,**  and 

*"Congres.  Inter,  de  Dermatol.  et  de  Syphiligr.,"  Paris,  1889; 
"Compt  rendu,"  p.  229. 

f  "Monatschr.  f.  praktische  Dermatologie,"  Bd.  xiv,  1892,  p.  485. 
J  "Archiv.  f.   Dermatol.  u.  Syphilol.,"  1897,  p.  263;   1897,  p.  41. 
§  "Centralbl.  f.  Bakt.,"  etc.,  1893,  Xm,  p.  743. 
§  "Med.  Moderne,"  Paris,  1893,  iv,  p.  735. 
**  "Ann.  de  Dermat.  et  de  Syphil.,"  Par.,  1894,  P-  272- 

442 


Cultivation  443 

Davis*  have  abundantly  confirmed  the  observations  of 
Ducrey  and  Unna,  and  proved  the  identity  of  the  two 
micro-organisms  and  their  specificity  for  the  disease. 

Morphology. — The  organism  is  commonly  described  as 
a  "  strep tobacillus."  It  is  very  small,  short,  and  ovoid  in 
shape,  and  occurs  habitually  in  longer  or  shorter  chains. 
Each  organism  measures  about  1.5  +  0.5  fi.  The  ends  are 
rounded  and  stain  deeply.  In  pure  cultures  long  undivided 
filaments,  at  least  twenty  times  as  long  as  the  individual 
bacilli,  are  not  uncommon.  There  seems  to  be  no  relation 
between  the  cells  and  the  bacilli.  As  a  rule,  they  are  free, 
sometimes  they  are  inclosed  in  leukocytes. 


•7     -*--•< 


^r;* 


Fig.  138. — Smear  of  pus  of  chancroid  of  penis  stained  with  carbol- 
fuchsin  and  briefly  decolorized  by  alcohol.  X  1500  (Davis).  (Photo- 
micrograph by  Mr.  L.  S.  Brown.) 

Staining. — The  organisms  are  somewhat  difficult  to  stain, 
as  they  do  not  retain  the  color  well,  giving  it  up  quickly 
when  washed.  They  do  not  stain  by  Gram's  method. 

Cultivation. — The  first  successful  isolation  and  cultiva- 
tion of  the  organism  seems  to  have  been  by  Benzancon, 
Griffon  and  Le  Sours  f  upon  a  culture-medium  consisting  of 
rabbits'  blood  i  part,  and  agar-agar  2  parts.  Davis  J  has 
been  equally  successful  in  cultivating  the  organism  upon  this 
medium.  His  method  was  as  follows: 

"Tubes  of  2  per  cent,  agar,  reaction  +  1.5,  were  melted 

*  " Jour.  Med.  Research,"  1893,  IX»  P-  4QI- 

t  ''Ann.  de  Dermat.  et  de  Syphiligr.,"  1901,  n,  p.  i. 

J  Loc.  cit. 


444  Chancroid 

and  mixed  with  fresh  rabbits'  blood  drawn  under  aseptic 
precautions,  in  the  proportion  of  two-thirds  agar  to  one- 
third  blood,  and  slanted  while  in  a  fluid  state.  At  a  later 
period  tubes  of  rabbits'  blood-serum  uncoagulated,  also 
rabbits'  blood  bouillon,  one-third  blood  to  two-thirds  bouil- 
lon, were  used,  and  gave  equally  satisfactory  results.  By 
employing  small  tubes  of  freshly  drawn  human  blood  pure 
cultures  were  obtained  in  several  instances  from  genital 
lesions  direct  without  any  special  cleansing  of  the  ulcerated 
surface.  This,  I  believe,  is  the  best  medium  for  obtaining 
cultures  from  a  source  open  to  contamination,  the  fresh 


Fig.  139. — Culture  from  ulceration  on  monkey  resulting  from  inocu- 
lation of  culture  from  a  case  of  chancroid  of  finger,  first  generation. 
Stained  with  carbol-fuchsin  and  briefly  decolorized  by  alcohol.  Cul- 
ture of  twenty-four-hours'  growth  in  rabbit's  bouillon.  X  1500  (Davis). 
(Photomicrograph  by  Mr.  L.  S.  Brown.) 

blood  apparently  inhibiting  to  a  certain  extent  the  growth 
of  extraneous  organisms." 

No  growth  takes  place  upon  ordinary  culture-media  under 
either  aerobic  or  anaerobic  conditions. 

Cultures  are  best  obtained  by  puncturing  an  unopened 
bubo  with  a  sterile  needle  and  planting  the  pus  directly  and 
immediately  upon  the  special  medium  which  should  have 
been  warmed  in  the  incubator  so  that  the  pus  is  not  chilled. 
In  this  way  pure  cultures  may  be  secured,  which  are  difficult 
to  get  from  the  soft  sore  itself. 

Colonies. — The  colonies  appear  upon  the  appropriate 
media  in  about  twenty-four  hours,  and  attain  their  complete 


Pathogenesis  445 

development  in  about  forty-eight  hours.  They  are  at  first 
round  bright  globules,  and  later  become  grayish  and  opaque. 
They  measure  i  to  2  mm.  in  diameter  and  never  become 
confluent.  They  are  difficult  to  pick  up  with  the  platinum 
wire,  tending  to  slide  over  the  smooth  surface  of  the  medium. 

Vital  Resistance. — The  organisms  seem  to  possess  little 
vitality,  their  life  in  artificial  culture  being  limited  to  a  few 
days.  Frequent  transplantation  enabled  Davis  to  carry 
them  on  to  the  eleventh  cultural  generation. 

Pathogenesis. — The  organism  is  pathogenic  for  man 
and  certain  monkeys  (macacus),  but  not  for  the  ordinary 
laboratory  animals.  The  organism  can  be  found  in  large 
numbers  in  both  the  genital  and  extragenital  chancroidal 
lesions,  and  usually  in  small  numbers  in  the  pus  from  chan- 
croidal buboes.  It  has  not  been  encountered  elsewhere. 
Lenglet*  isolated  the  organism  in  pure  culture,  and  by 
inoculation  with  his  cultures  reproduced  the  lesions  in  man. 

*"Bull.  Med.,"  1898,  p.  1051;  "Ann.  de  Dermatol.  et  de  Syph.," 
1901,  t.  n.  p.  209. 


CHAPTER  X. 

ACUTE  CONTAGIOUS  CONJUNCTIVITIS. 

THE  KOCH-WEEKS  BACILLUS. 

General  Characteristics. — A  minute,  slender  bacillus,  non-motile, 
non-flagellated,  non-sporogenous,  non-liquefying,  non-chromogenic, 
aerobic,  and  optionally  anaerobic,  staining  by  the  ordinary  methods 
but  not  by  Gram's  method,  susceptible  of  cultivation  upon  special 
media  only,  and  specific  for  acute  contagious  conjunctivitis. 

Acute  contagious  conjunctivitis  is  a  common  and  world- 
wide affection,  sometimes  called  "pink  eye,"  and  sometimes 
erroneously  called  catarrhal  conjunctivitis.  All  its  char- 
acteristics, and  especially  its  contagiousness,  point  to  its 
being  a  specific  disease  due  to  a  specific  cause,  and  thus 
entirely  different  from  ordinary  non-specific  catarrh. 

Specific  Micro=organism. — The  first  bacteriologic  inves- 
tigation of  acute  contagious  conjunctivitis  was  made  by 
Robert  Koch,*  when  in  Egypt  investigating  a  cholera 
epidemic.  While  in  Alexandria  he  examined  the  secretions 
from  50  cases  of  conjunctivitis,  finding  the  gonococcus, 
or  an  organism  closely  resembling  it.  In  a  less  severe  form 
of  the  disease,  however,  he  found  a  peculiar  small  bacillus. 
He  seemed  satisfied  with  this  observation,  or  had  no  time  to 
pursue  the  matter  farther,  for  no  cultivation  or  other  ex- 
periments are  mentioned. 

The  organism  was  observed  from  time  to  time,  but  no 
serious  consideration  seems  to  have  been  devoted  to  it  until 
Weeks  f  published  an  account  of  what  seemed  to  be  the  identi- 
cal organism,  which  he  not  only  observed,  but  alsp  cultivated, 
and  eventually  successfully  inoculated  into  the  human  con- 
junctiva. In  the  same  year  Kartulis|  in  Alexandria  suc- 
ceeded in  cultivating  the  same  organism.  In  1894  Morax 
published  a  brochure  in  Paris  in  which  he  says  that  "the 
disease  [which  he  describes  under  the  name  of  acute  con- 

*  "Wiener  klin.  Wochenschrift,"  1883,  p.  1550. 
t  "N.  Y.  Med.  Record,"  May  21,  1887. 
t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1887,  p.  289. 
446 


Staining  447 

junctivitis]  is  characterized  by  the  constant  presence  in  the 
conjunctival  secretions  of  a  small  bacillus  seen  for  the  first 
time  by  Koch,  but  studied  some  years  later  by  Weeks,  and 
now  known  as  the  bacillus  of  Weeks." 

Further  descriptive  and  clinical  information  can  be  found 
in  a  paper  by  Weeks,  "The  Status  of  our  Knowledge  of  the 
J^tiological  Factor  in  Acute  Contagious  Conjunctivitis."* 

Morphology. — The  organism  is  very  tiny  and  is  said  to 
bear  some  resemblance  to  the  bacillus  of  mouse-septicemia. 
It  measures  i  to  2  X  0.25  ^  (Weeks).  The  length  is  more 
constant  in  individuals  found  in  the  pus  than  those  taken 


Fig.  140. — The  Koch- Weeks  bacillus  in  conjunctival  secretion.     Magni- 
fied 1000  diameters  (Rymowitsch  and  Matschinsky). 


from  cultures.  In  cultures  the  organisms  are  longer  and 
more  slender.  Involution  forms  of  considerable  length  and 
of  irregular  shape  also  occur.  No  spores  are  observed.  The 
organism  has  no  flagella  and  is  not  motile. 

Staining. — Weeks  found  that  the  organism  stained  well 
with  watery  solutions  of  methylene-blue,  basic  fuchsin,  or 
gentian  violet.  The  color  is  fainter  than  that  of  the  nuclei 
of  the  associated  pus-corpuscles,  and  is  much  less  intense  in 
old  than  in  fresh  cultures.  It  is  readily  given  up  when 
treated  with  alcohol  or  acids.  Morax  found  that  the  bacilli 
did  not  retain  the  color  in  Gram's  method. 

''New  York  Eye  and  Ear  Infirmary  Reports,"  vol.  in,  Part  i, 
Jan.,  1895,  p.  24. 


448  Acute  Contagious  Conjunctivitis 

Cultivation. — The  organism  refuses  to  grow  upon  any  of 
the  ordinary  culture-media.  Weeks  found,  however,  that  if 
the  percentage  of  agar-agar  used  was  reduced  to  0.5  per 
cent.,  growths  could  be  secured  by  incubation  at  37°  C.,  and 
successful  transplantations  carried  on  to  the  sixteenth  gen- 
eration. Abundant  moisture  was  essential.  The  method 
of  isolation  adopted  by  Weeks  was  as  follows: 

"The  conjunctival  sacs  were  thoroughly  washed  with  clean  water, 
removing  the  secretion  present  by  means  of  absorbent  cotton.  The 
patient  was  then  directed  to  keep  the  eyes  closed.  After  five  or  ten 
minutes  had  elapsed,  the  eyes  were  opened,  and  the  secretion  that  had 
formed,  lying  at  the  bottom  of  the  cul-de-sac,  was  removed  by  means 
of  a  sterilized  platinum  rod  and  transferred  to  the  surface  of  the  agar. 
The  mass  of  tenacious  secretion  was  drawn  over  the  surface  of  the 
organ  and  left  there,  the  platinum  being  thrust  into  the  agar  two  or 
three  times  before  removal." 

At  the  end  of  forty-eight  hours  a  slight  haziness  appears 
along  the  path  of  the  wire,  and  on  the  surface  of  the  agar 
a  very  small  patch  is  noticeable ;  this  is  of  a  pearly  color  and 
possesses  a  glistening  surface.  By  the  formation  of  small 
concentric  colonies  the  growth  extends  for  a  short  distance. 
At  the  end  of  the  fourth  or  fifth  day  the  growth  ceases  to 
advance;  it  is  never  abundant.  The  culture  dies  in  from 
one  to  three  weeks. 

Pathogenesis. — Both  Weeks  and  Morax  have  tested  the 
organism  for  pathogenic  activity,  and  in  every  case  in  which 
pure  cultures  of  it  were  placed  upon  the  human  conjunctiva, 
typical  attacks  of  the  acute  conjunctivitis  resulted.  The 
organism  fails  to  infect  any  of  the  lower  animals. 

Association. — Both  Weeks  and  Morax  found  the  organ- 
ism in  intimate  association  with  a  larger  club-shaped  bacillus, 
which  was  regarded  as  the  pseudodiphtheria  bacillus.  It 
seems  to  be  of  no  pathogenic  significance. 

THE  MORAX-AXENFELD  BACILLUS. 

In  1896  Morax*  found  a  new  bacillus  in  certain  cases  of 
epidemic  subacute  conjunctivitis.  Immediately  afterward 
Axenfeldf  presented  to  a  congress  in  Heidelberg  cultures  of 
the  same  bacillus  that  he  had  isolated  from  5 1  cases  of  what 
he  called  "  diplobacillenconjunctivitis"  that  occurred  a  few 
months  before  as  an  epidemic  in  Marburg.  De  Schweinitz 

*  "Ann.de  1'Inst.  Pasteur,"  June,  1896;  "Ann.  d'Oculist,"  Jan.,  1897. 
f  "Heidelberg,  Congress,"  1896;  "Centralbl.  f.  Bakt.,"  etc.,  1897,  xxi. 


Cultivation  449 

and  Veasy,*  Alt,f  and  others  found  the  same  diplobacillus  in 
America,  and  many  others  confirmed  the  observations  in 
various  parts  of  Europe.  It  has  also  been  found  in  Egypt. 
There  is  no  doubt,  therefore,  but  that  this  is  a  widely  distrib- 
uted organism.  Morax  produced  the  disease  by  placing  a 
pure  culture  of  the  organism  upon  the  human  conjunctiva. 
He  was  unable  to  infect  any  of  the  lower  animals. 

In  this  subacute  form  of  conjunctivitis  there  is  very  little 
secretion,  and  to  secure  the  micro-organism  either  for  micro- 
scopic examination  or  for  cultivation  recourse  must  be  had  to 
minute  flakes  of  grayish  mucus  that  collect  upon  the  caruncle. 


Fig.  141. — The  Morax-Axenfeld  diplobacillus  of  conjunctivitis.    Magni- 
fied 1000  diameters  (Rymowitsch  and  Matschinsky. 

Morphology. — The  bacillus  is  small,  commonly  occurs  in 
pairs  or  chains.  It  measures  approximately  2  [i  in  length. 
It  is  not  motile,  has  no  flagella,  and  forms  no  spores.  It  is 
somewhat  pleomorphous.  Involution  forms  soon  appear  in 
artificial  cultures. 

Staining. — The  organism  stains  by  ordinary  methods,  but 
does  not  stain  by  Gram's  method. 

Cultivation. — The  organism  grows  only  upon  alkaline 
blood-serum  or  upon  culture-media  containing  blood-serum. 
Morax  made  his  original  observation  by  using  L°ffler's 
blood-serum  mixture.  The  colonies  appear  in  twenty-four 
hours  at  37°  C.  The  blood-serum  is  almost  immediately 

*  "  Ophthalmological  Record,"  1899. 
t  "Amer.  Jour,  of  Ophthalmology,"  1898,  p.  171. 
29 


45°  Acute  Contagious  Conjunctivitis 

liquefied,  so  that  the  growing  colonies  appear  to  be  sinking  into 
the  medium  after  thirty-six  hours.  The  entire  tube  of  medium 
may  eventually  be  liquefied. 

Upon  agar-agar  containing  serum,  grayish-white  colonies 
of  small  size,  resembling  colonies  of  gonococci,  are  formed. 
Growth  is  slow.  Bouillon  is  slowly  clouded. 

Pathogenesis. — The  pathogenic  and  specific  nature  of 
the  diplobacillus  was  made  clear  by  Morax,  who  produced  the 
disease  in  man  by  placing  a  pure  culture  upon  the  human  con- 
junctiva. 

ZUR  NEDDEN'S  BACILLUS. 

This  bacillus  was  the  only  organism  that  Haupt*  was  able 
to  isolate  from  a  neuroparalytic  with  confluent  peripheral 
ulcer ations  of  the  cornea.  It  seemed  to  be  identical  with  an 
organism  that  zur  Nedden  had  found  previously  in  a  case  of 
corneal  ulceration  in  the  clinic  at  Bonn. 

Morphology. — It  is  a  tiny  bacillus,  less  than  i  u  in  length, 
slightly  curved,  generally  single,  but  sometimes  in  pairs  and 
short  chains.  It  is  not  motile,  has  no  flagella,  forms  no  spores. 

Staining. — It  stains  ordinarily,  but  not  by  Gram's 
method. 

Cultivation. — It  is  easily  cultivated  upon  the  ordinary 
laboratory  media,  the  cultures  being  without  characteristic 
peculiarities.  Gelatin  is  not  liquefied.  Milk  is  coagulated. 
Acid  but  no  gas  is  formed  in  glucose  media.  A  thick  yellow- 
ish growth  appears  upon  potato.  No  indol  is  formed. 

Pathogenesis. — Corneal  ulcers  were  formed  in  a  guinea- 
pig  after  artificial  implantation  in  the  corneal  tissue. 

MISCELLANEOUS  ORGANISMS  IN  CONJUNCTIVITIS. 

In  addition  to  the  foregoing  organisms,  others  not  infre- 
quently make  their  appearance  as  excitants  of  conjunctivitis. 
The  most  frequent  of  these  is  the  pneumococcus,  the  most 
dangerous,  the  gonococcus.  The  former  produce  a  severe 
conjunctivitis,  with  the  formation  of  a  false  membrane,  the 
latter  the  well-known  blenorrhea  and  ophthalmia  neonatorum. 
Streptococci,  diphtheria  bacilli,  staphylococci,  meningococci, 
colon  bacilli,  Bacillus  pneumonia  (Friedlander),  and  other 
organisms  have  been  found  and  appear  to  be  responsible  for 
conjunctivitis. 

*"  Inaugural  Dissertation,"  Bonn,  1902. 


CHAPTER  XI. 

DIPHTHERIA. 

BACILLUS  DIPHTHERIA  (KLKB 

General  Characteristics. — A  non-motile,  non-flagellate,  non-spor- 
ogenous,  non-chromogenic,  non-liquefying,  aerobic,  purely  parasitic, 
pathogenic,  toxicogenic  bacillus,  cultivable  upon  the  ordinary  culture 
media,  staining  by  the  ordinary  methods  and  by  Gram's  method. 

In  1883  Klebs  *  demonstrated  the  presence  of  a  bacillus 
in  the  pseudo-membranes  upon  the  fauces  of  patients 
suffering  from  diphtheria,  but  it  was  not  until  1884  that 
Loffler  f  succeeded  in  isolating  and  cultivating  it.  The 
organism  is  now  known  by  both  their  names,  and  called  the 
Klebs-Loffler  bacillus. 

Morphology. — The  bacillus  is  about  the  length  of  the 
tubercle  bacillus  (1.5-6.5  /*),  but  about  twice  its  diameter 
(0.4-1.0  /*),  has  a  slight  curve  similar  to  that  which  char- 
acterizes the  tubercle  bacillus,  and  has  rounded  and  usu- 
ally clubbed  ends.  It  does  not  form  chains,  though 
two,  three,  and  rarely  four  individuals  may  be  found  con- 
joined; usually  the  individuals  are  separate  from  one  an- 
other. The  bacillus  is  peculiar  in  its  pleomorphism,  for 
among  the  well-formed  individuals  which  abound  in  fresh 
cultures  a  large  number  of  peculiar  organisms  are  to  be 
found,  much  larger  than  normal,  some  with  one  end  en- 
larged and  club  shaped,  some  greatly  elongated,  with  both 
ends  similarly  and  irregularly  expanded.  These  bizarre 
forms  probably  represent  an  involution  form  of  the  organ- 
ism, for,  while  present  in  perfectly  fresh  cultures,  they 
are  much  more  abundant  in  old  cultures  where  scarcely  a 
single  well-formed  bacillus  can  be  found.  Distinct  polar 

*  "  Verhandlungen  des  Congresses  fur  innere  Med.,"  1883. 
f  "  Mittheilungen  aus  dem  kaiserlichen  Gesundheitsamte,"  2. 

45i 


45 2  Diphtheria 

granules  can  be  denned  at  the  ends  of  the  bacilli.  Occa- 
sional branched  forms  are  observed,  and  the  diphtheria 
bacillus  probably  belongs  to  the  higher  bacteria,  though 
Abbott  and  Gildersleeve*  do  not  regard  branching  as  a  phase 
of  the  normal  development  of  the  organism,  do  not  find  it. 
common  upon  the  standard  culture  media,  and  so  do  not 
think  that  it  is  properly  classified  elsewhere  than  among  the 
bacilli. 

No  flagella  have  been  demonstrated  upon  the  bacillus, 
and  it  is  non-motile.  It  is  almost  purely  aerobic. 

The  involution  of  the  diphtheria  bacillus  seems  to  occur 
in  proportion  to  the  rapidity  of  its  growth.  Upon  Loffler's 
serum  mixture,  which  seems  best  adapted  for  its  cultivation, 
the  involution  of  the  organism  takes  place  with  great 
rapidity,  so  that  large  clubbed  organisms  and  large  or- 
ganisms with  polar  granules  are  very  common.  On  the 
other  hand,  upon  agar  and  glycerin  agar-agar,  where  the 
organism  grows  very  slowly,  it  usually  appears  in  the  form 
of  short  spindle  and  lancet  shapes.  So  different  are  these 
forms  that  a  beginner  would  certainly  fail  to  recognize  them 
as  identical.  The  small  short  forms  also  stain  much  more 
uniformly  than  the  large  club-shaped  bacilli. 

Wesbrook  f  has  established  certain  morphologic  types  of 
the  bacillus  (see  illustration),  and  from  the  appearances 
presented  draws  conclusions  regarding  their  virulence, 
which  are  confirmed  by  Gorham,J  but  disputed  by  Denny.  § 
The  rapidly  growing  bacilli  with  clubbed  ends  and  polar 
granules  are  supposed  to  be  virulent  forms;  the  slowly 
growing,  uniformly  staining  forms,  non-virulent  bacilli. 
Park  and  Denny  believe  that  the  uniformly  staining  bacil- 
lus, when  it  develops  in  blood-serum  cultures,  is  the  pseudo- 
diphtheria  bacillus,  an  entirely  different  organism. 

Staining. — The  bacillus  can  readily  be  stained  with  aque- 
ous solutions  of  the  analin  colors,  but  more  beautifully 
and  characteristically  with  Loffler's  alkaline  methylene- 
blue: 

*  "Centralbl.  f.  Bakt.,"  etc.,  Dec.  18,  1903,  Bd.  xxxv,  No.  3. 

t  "Trans.  Assoc.  Amer.  Phys.,"  1900,  and  "Trans,  of  the  Amer. 
Public  Health  Assoc.,"  1900. 

t  "Journal  of  Medical  Research,"  N.  S.,  vol.  i,  p.  201,  1901. 

§  American  Public  Health  Association  (New  Orleans)  Meeting, 
1902. 


Staining  453 

Saturated  alcoholic  solution  of  methylene-blue . .     30 
1  : 10,000  aqueous  solution  of  caustic  potash   .  .    100 

Emery  prefers  Hanson's  borax  methylene-blue.  A  stock 
solution  which  keeps  well  is  prepared  by  dissolving  2  grams 
of  methylene-blue  and  5  grams  of  borax  in  100  c.c.  of  water. 
This  is  diluted  with  from  five  to  ten  times  its  volume  of 
water  for  ordinary  use.  An  aqueous  solution  of  dahlia 
is  recommended  by  Roux. 

The  Neisser  method  of  staining  the  diphtheria  bacillus, 
which  met  with  a  very  cordial  reception,  is  as  follows: 

The  prepared  cover-glass  is  immersed  for  from  two  to 
three  seconds  in 


Alcohol  (96  per  cent.) 20  parts 

Methylene-blue 1  part 

Distilled  water 950  parts 

Acetic  acid  (glacial) 50     " 


Then  for  three  to  five  seconds  in 

Bismarck  brown 1  part 

Boiling  distilled  water 500  parts 

The  true  diphtheria  bacilli  appear  brown,  with  a  dark  blue 
body  at  one  or  both  ends;  the  pseudo-diphtheria  .bacilli 
usually  exhibit  no  polar  bodies. 

Park  *  in  his  large  experience  found  that  neither  the 
Neisser  nor  the  Roux  stain  gave  any  more  information 
concerning  the  virulence  of  the  bacilli  than  the  Loffler 
alkaline  methylene-blue. 

When  cover-glass  preparations  are  stained  with  these 
solutions,  the  bizarre  forms  already  mentioned  are  par- 
ticularly obvious,  and  the  contrast  between  the  polar 
granules,  which  color  intensely,  and  the  cytoplasm  of  the 
bacillus,  which  tinges  slightly,  is  marked.  Through  good 
lenses  the  organisms  are  always  distinct  bacilli,  notwith- 
standing the  fact  that  the  ends  stain  more  deeply  than  the 
centers,  and  it  is  only  through  poor  lenses  that  the  organ- 
isms can  be  mistaken  for  diplococci. 

The  bacilli  stain  well  by  Gram's  method,  which  is  ex- 
cellent for  their  definition  in  sections  of  tissue,  though 

*  "Bacteriology  in  Medicine  and  Surgery,"  1900. 


454  Diphtheria 

Welch  and  Abbott  found  that  Weigert's  fibrin  method  and 
picrocarmin  gave  the  most  beautiful  results. 

Cultivation. — The  diphtheria  bacillus  grows  readily  upon 
all  the  ordinary  media,  and  is  very  easy  to  obtain  in  pure 
culture,  plates  not  being  necessary.  Material  from  the  in- 
fected throat  can  be  taken  with  a  swab  or  platinum  loop 
and  spread  upon  the  surface  of  several  successive  tubes 
of  Loffler's  blood-serum  media.  Upon  the  first  a  confluent 
growth  of  the  bacillus  usually  occurs;  but  upon  the  third, 
scattered  colonies  suitable  for  transplantation  can  usually 
be  found. 

Loftier  has  shown  that  the  addition  of  a  small  amount 
of  glucose  to  the  culture  medium  increases  the  rapidity  of 
growth,  and  suggests  a  special  medium  which  bears  his 
name — Loffler's  blood-serum  mixture: 


Blood-serum  .  % 3 

Ordinary  bouillon  +  1  per  cent,  of  glucose ....    1 


This  mixture  is  filled  into  tubes,  coagulated,  and  sterilized 
like  blood-serum,  and  is  one  of  the  best  known  media  to  be 
used  in  connection  with  the  study  of  diphtheria. 

The  studies  of  Michel  *  have  shown  that  the  development 
of  the  culture  is  much  more  luxuriant  and  rapid  when 
horses'  serum  instead  of  beef  or  calves'  serum  is  used. 
Horse's  blood  can  easily  be  secured  by  the  introduction  of 
a  trocar  into  the  jugular  vein ;  5  liters  of  it  can  be  withdrawn 
without  causing  the  animal  inconvenience  or  symptoms  of 
weakness. 

Westbrook  suggested  that  the  addition  of  a  small  amount 
of  glycerin  to  the  preparation  of  blood-serum  would  prevent 
it  from  drying  so  rapidly  as  usual  and  would  have  the  added 
advantage  of  preventing  the  growth  of  certain  varieties 
of  bacteria  not  desired.  Duboisf  carried  out  a  series  of 
observations  upon  this  question  and  found  that  3  to  5 
per  cent,  of  glycerin  makes  a  very  valuable  addition,  as 
the  diphtheria  bacilli  grow  very  rapidly  and  almost  in 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Sept.  24,  1897,  Bd.  xxn, 
Nos.  10  and  u. 

t  "Seventeenth  Annual  Report  of  the  Department  of  Health  and 
Charities,"  Indianapolis,  Ind.,  1907. 


Fig.  142. — Bacillus  diphtheriae,  five  hours  Fig.  143. — Bacillus  diphtheriae,  same  cul- 

at  36°  C.    This  shows  only  solid  staining        ture,  eight  hours  at  36°  C.    This  also  shows 


forms. 


solid  forms,  many  of  them  with  parallel  ar- 
rangement. 


\/i  <*'* 

N  '  »  %** 

.'•-.'**V'    '• 
' YX  *T*  -VJ 

A    .  J          +-~^' 


-':'fr'\?~4^ 
,/^lAr/r 

*  fc'^y  N \ 

m  •>    '-arVrA 


Fig.  144. — Bacillus  diphtheriae,  same  cul- 
ture, twelve  hours  at  36°  C.  The  bacilli 
stain  faintly  at  their  ends,  and  in  some  small 
granules  are  seen  at  the  tip  of  the  faintly 
stained  portions. 


Fig.  145. — Bacillus  diphtheriae,  same  cul- 
ture, fifteen  hours  at  36°  C.  The  bacilli 
stain  more  unevenly  and  the  granules  are 
larger. 


r^-  ^ 


v-^ 


Fig.  146. — Bacillus  diphtheriae,  same  cul- 
ture, twenty-four  hours  at  36°  C.  This 
shows  clubbed  and  barred  forms  as  well  as 
granular  forms.  At  the  lower  part  of  the 
field  is  a  paired  form  which  shows  the  char- 
acteristic clubbing  of  the  distal  ends. 

(Photomicrographs  by  Mr.  Louis  Brown. 

All  of  the  preparations  were  made  from  growth  on  blood-serum.) 
"  Jour,  of  Med.  Research.") 

455 


Fig.  147.  —  Bacillus  diphtheriae,  forty- 
eight  hours  at  36°  C.  This  is  the  same 
bacillus  as  in  the  preceding  figures,  but  from 
a  culture  where  the  colonies  were  discrete. 
It  shows  long  filamentous  forms. 


The  magnification  is  the  same  in  all  —  X  2000. 
(Francis  P.  Denny,  in 


456 


Diphtheria 


pure  culture  upon  the  blood-serum  mixture  to  which  it  is 
added. 

The  impossibility  of 
making  an  accurate 
diagnosis  of  diph- 
theria without  a  bac- 
teriologic  examination 
has  caused  many  pri- 
vate physicians  and 
many  medical  socie- 
ties and  boards  of 
health  to  equip  labor- 
atories where  bacte- 
riologic  examinations 
can  be  made.  The 
method  requires  some 
apparatus,  though  a 
competent  bacteriolo- 
gist can  often  make 
shift  with  a  bake- 
oven,  a  wash-boiler, 
and  other  household 
furniture,  instead  of 
the  regular  sterilizers 
and  incubators,  which 
are  expensive. 

Bacteriologic  Di- 
agnosis.— When  it  is 
desired  to  make  a 
bacteriologic  diagno- 
sis in  suspected  diph- 
theria, or  to  secure  the 

bacillus  in  pure  culture,  a  sterile  platinum  wire  having 
a  small  loop  at  the  end,  or  a  swab  made  by  wrapping  a 
little  absorbent  cotton  about  the  end  of  a  piece  of  wire 
and  carefully  sterilizing  it  in  a  test-tube,  is  introduced 
into  the  throat  and  touched  to  the  false  membrane,  after 
which  it  is  carefully  smeared  over  the  surface  of  at  least 
three  of  the  blood-serum  mixture  tubes,  without  either 
again  touching  the  throat  or  being  sterilized.  The  tubes 
thus  inoculated  are  stood  away  in  an  incubating  oven  at 
the  temperature  of  37°  C.  for  twelve  hours,  then  exam- 


Fig.  148. — The  Providence  Health  De- 
partment outfit  for  diphtheria  diagnosis, 
consisting  of  a  pasteboard  box  containing  a 
swab-tube  and  a  serum-tube,  both  with 
etched  surface  on  which  to  write  the  name 
and  address  of  patients,  etc. 


Cultivation 


457 


istf 


ined.  If  the  diphtheria  bacillus  be  present,  a  smeary, 
yellowish- white  layer  will  be  present  upon  the  first  tube,  a 
similar  layer  with  outlying  colonies  on  the  second  tube, 
while  the  third  tube  will  show  rather 
large,  isolated,  whitish  or  slightly 
yellowish,  smooth  colonies.  The 
colonies  may  be  china-white  in  ap- 
pearance. These  colonies,  if  found 
by  microscopic  examination  to  be 
made  up  of  diphtheria  bacilli,  will 
confirm  the  diagnosis  of  diphtheria, 
and  will  at  the  same  time  give 
pure  cultures  of  the  bacillus  when 
transplanted.  There  are  very  few 
other  bacilli  that  grow  so  rapidly 
upon  Loffler's  mixture,  and  scarcely 
any  other  is  found  in  the  throat. 

When  no  tubes  of  the  blood- 
serum  mixture  are  at  hand,  the 
swab  can  be  returned  to  its  tube 
after  having  been  wiped  over  the 
throat  of  the  patient,  and  can  be 
shipped  to  the  nearest  laboratory. 

When  an  early  diagnosis  is  re- 
quired, Ohlmacher  recommends 
that  the  microscopic  examination 
of  the  still  invisible  growth  be  made 
in  five  hours.  A  platinum  loop  is 
rubbed  over  the  inoculated  surface ; 
the  small  amount  of  material  thus 
secured  is  mixed  with  distilled 
water,  spread  on  a  cover-glass, 
dried,  fixed,  stained  with  methy- 
lene-blue,  and  examined.  An  abun- 
dance of  the  organisms  are  usually 
found  and  valuable  time  is  saved  preparatory  to  the  use  of 
the  antitoxin. 

The  presence  of  diphtheria  bacilli  in  material  taken  from 
the  throat  does  not  necessarily  mean  that  the  person  has 
diphtheria.  Virulent  bacilli  can  occasionally  be  discovered 
in  the  throats  of  healthy  persons  who  have  knowingly  or 
unknowingly  come  in  contact  with  the  disease,  but  whose 


Fig ,  1 49 . —  Sterilized 
test-tube  and  swab  for 
collecting  pus  and  fluids 
for  bacteriologic  examina- 
tion (Warren). 


458 


Diphtheria 


vital  resistance  is  such  that  the  bacilli  grow  scantily  without 
producing  disease  of  the  throat.  The  bacteriologic  examin- 
ation is,  therefore,  only  an  adjunct  to  the  clinical  diagnosis, 
and  must  never  be  taken  as  positive  in  itself. 


Fig.  150. — Diphtheria  bacilli  (from  photographs  taken  by  Prof.  E. 
K.  Dunham,  Carnegie  Laboratory,  New  York):  a,  Pseudobacillus ; 
b,  true  bacillus;  c,  pseudobacillus. 

Gelatin. — Gelatin  is  not  an  appropriate  medium  for  the 
cultivation  of  the  bacillus.  Upon  the  surface  of  gelatin 
plates  the  colonies  attain  but  a  small  size  and  appear  to 
the  naked  eye  as  whitish  points  with  smooth  contents  and 
regular,  though  sometimes  indented,  borders.  Under  the 
microscope  they  appear  granular  and  yellowish-brown,  with 
irregular  borders  (Fig.  151).  The  growth  in  gelatin  punc- 
ture is  characterized  by  the  occurrence  of  small  spheric 
colonies  along  the  line  of  inoculation.  The  gelatin  is  not 
liquefied. 


Cultivation  459 

Agar-agar. — Upon  agar-agar  and  glycerin  agar-agar  the 
colonies  are  slower  to  develop,  larger,  more  translucent, 
without  the  yellowish-white  or  china-white  color  of  the 
blood-serum  cultures,  and  are  more  or  less  distinctly  divided 
into  a  small  elevated  center  and  a  flat  surrounding  zone 
with  indented  edges,  and  a  radiated  appearance.  When 
transplantations  are  made  from  blood-serum  to  agar-agar, 
the  resulting  growth  is  usually  meager,  but  the  oftener  the 
organism  is  transplanted  to  fresh  agar-agar,  the  more  luxu- 
riant its  growth  becomes. 

Bouillon. — When  planted  in  bouillon  a  distinct,  whitish, 


Fig.  151. — Bacillus  diphtheriae ;   colony  twenty-four   hours  old,  upon 
agar-agar.     X  100  (Frankel  and  Pfeiffer). 


granular  pellicle  forms  upon  the  surface  of  the  medium. 
The  pellicle  appears  quite  uniform  when  the  flask  is  un- 
disturbed, but  it  is  so  brittle  that  it  at  once  falls  to  pieces  if 
the  flask  be  moved,  the  minute  fragments  slowly  sedimenting 
and  forming  a  miniature  snow-storm  in  the  flask  or  tube. 
The  organism  at  times  also  causes  a  diffuse  cloudiness  of  the 
medium,  but,  not  being  motile,  soon  settles  to  the  bottom 
in  the  form  of  a  flocculent  precipitate  which  has  a  tendency 
to  cling  to  the  sides  of  the  glass,  and  leave  the  bouillon 
clear. 


460  Diphtheria 

Spronk  *  found  that  the  characteristics  of  the  growth  of 
the  diphtheria  bacillus  in  bouillon,  as  well  as  the  amount 
of  toxin  produced,  vary  according  to  the  amount  of  glucose 
in  the  bouillon. 

Zinnof  found  that  digested  brain  added  to  the  culture 
bouillon  greatly  facilitated  the  growth  of  diphtheria  and 
tetanus  bacilli  and  increased  the  toxin- product  ion. 

Blood-serum. — The  bacillus  grows  similarly  upon  blood- 
serum  and  Loffler's  mixture. 

Potato. — Upon  potato  it  develops  only  when  the  reaction 
is  alkaline.  The  potato  growth  is  not  characteristic. 

Milk. — Milk  is  an  excellent  medium  for  the  cultivation  of 
Bacillus  diphtherise,  and  is  possibly  at  times  a  medium  of 
infection.  Litmus  milk  is  useful  for  detecting  the  changes 
of  reaction  brought  about  by  the  alkalinity,  which  at  first 
favors  the  development  of  the  bacillus,  being  soon  replaced 
by  acidity.  When  the  culture  becomes  old,  the  reaction 
again  becomes  strongly  alkaline.  This  variation  in  reaction 
seems  to  depend  entirely  on  the  transformation  of  the  sugars. 

Vital  Resistance. — The  diphtheria  bacillus  does  not 
form  spores.  It  possesses  very  little  vital  resistance  and 
is  delicate  in  its  thermic  sensitivity.  LofHer  found  that  it 
could  not  endure  a  temperature  of  60°  C.,  and  Abbott  has 
shown  that  a  temperature  of  58°  C.  is  fatal  to  it  in  ten 
minutes.  The  organism  can  sometimes  be  kept  alive  for 
several  weeks  after  being  dried  upon  shreds  of  silk  or  when 
surrounded  by  dried  diphtheria  membrane. 

Metabolic  Products. — The  earliest  researches  upon  the 
nature  of  the  poisonous  products  of  the  diphtheria  bacillus 
seem  to  have  been  made  in  1887  by  Loffler,  {  who  came  to 
the  conclusion  that  they  belonged  to  the  enzymes.  The 
credit  of  removing  the  bacteria  from  the  culture  by  filtration 
through  porcelain  and  the  demonstration  of  the  soluble 
poison  in  the  filtrate  belongs  to  Roux  and  Yersin.§  Toxic 
bouillon  prepared  in  this  manner  was  found  to  cause  serous 
effusions  into  the  pleural  cavities,  acute  inflammation  of 
the  kidneys,  fatty  degeneration  of  the  liver,  and  edema 
of  the  tissue  into  which  the  injection  was  made.  In  some 
cases  palsy  subsequently  made  its  appearance,  usually  in  the 

*  "Ann.  del'Inst.  Pasteur,"  Oct.  25,  1895,  vol.  ix,  No.  10,  p.  758. 
t  "Centralbl.  f.  Bakt.,"  Jan.  4,  1902,  xxxi,  No.  2,  p.  42. 
J  "Centralbl.  f.  Bakt.,"  etc.,  1887,  n,  p.  105. 
§  "  Ann.  de  1'Inst.  Pasteur,"  1888-1889. 


Metabolic  Products  461 

hind  quarters.  The  effect  of  the  poison  was  slow  and  death 
took  place  days  or  weeks  after  injection,  sometimes  being 
preceded  by  marked  emaciation.  Temperatures  of  58°  C. 
lessened  the  activity  of  the  toxin  and  temperatures  of  100° 
C.  destroyed  it.  It  was  precipitated  by  absolute  alcohol 
and  mechanically  carried  down  by  calcium  chlorid.  Brieger 
and  Frankel  *  confirmed  the  work  of  Roux  and  Yersin,  and 
concluded  that  the  poison  was  a  toxalbumin.  Tangl  f 
was  able  to  extract  the  toxin  from  a  fragment  of  diphtheria 
pseudo-membrane  macerated  in  water. 

The  nature  of  the  diphtheria  toxin  has  been  studied  by 
Khrlich  {  and  found  to  be  extremely  complex.  As  it 
exists  in  cultures  it  is  composed  of  equal  parts  of  toxin  and 
toxoid.  Of  these,  the  former  is  poisonous,  the  latter  harm- 
less for  animals — or  at  least  not  fatal  to  them.  The  toxoids 
have  equal  or  greater  affinity  for  combining  with  antitoxin 
than  the  toxin  and  cause  confusion  in  testing  the  unit  value 
or  strength  of  the  antitoxin.  In  old  or  heated  toxin  all 
of  the  toxin  molecules  become  changed  into  toxons  or  tox- 
oids and  the  poisonous  quality  is  lost  though  the  power  of 
combining  with  antitoxin  remains. 

The  toxin  is  intensely  poisonous,  and  a  filtered  bouillon 
containing  it  may  be  fatal  to  a  3oo-gram  guinea-pig  in  doses 
of  only  0.0005  c-c-  It  is  thought  not  to  be  an  albuminous 
substance,  as  it  can  be  elaborated  by  the  bacilli  when 
grown  in  non-albuminous  urine,  or,  as  suggested  by  Uschin- 
sky,  in  non-albuminous  solutions  whose  principal  ingredient 
is  asparagin.  The  toxic  value  of  the  cultures  is  greatest 
in  the  second  week. 

This  soluble  toxin  so  well  known  in  bouillon  cultures  is 
probably  only  one  of  the  poisonous  substances  produced  by 
the  bacillus.  An  intracellular,  insoluble  toxic  product 
seems  to  have  been  discovered  by  Rist,§  who  found  it  in 
the  bodies  of  dried  bacilli,  and  observed  that  it  was  not 
neutralized  by  the  antitoxin. 

Palmirski  and  Orlowski||  assert  that  the  bacillus  pro- 
duces indol,  but  only  after  the  third  week.  Smith,**  how- 

*"  Berliner  klin.  Wochenschrift,"  1890,  11-12. 
f  "Centralbl.  f.  Bakt.,"  etc.,  Bd.  xi,  p.  379. 
J  "Klinisches  Jahrbuch,"  1897. 
§  "Soc.  de  Biol.  Paris,"  1903,  No.  25. 
||  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  March,  1895. 
**  "Jour.  Exp.  Med.,"  Sept.,  1897,  vol.  n,  No.  5,  p.  546. 


462  Diphtheria 

ever,  found  that    when    the    diphtheria   bacillus   grew    in 
dextrose-free  bouillon  no  indol  was  produced. 

The  acidity  of  the  culture  media  depends  upon  the 
formation  of  lactic  acid. 

Pathogenesis. — Diphtheria  in  man  is  characterized  by  a 
pseudo-membranous  inflammation  of  the  mucous  mem- 
branes, particularly  of  the  fauces,  though  it  may  occur  upon 
other  parts  of  the  body  and  is  not  infrequent  in  the  nose,  in 
the  mouth,  upon  the  genital  organs,  or  upon  wounds.  Wil- 
liams *  has  reported  a  case  of  diphtheria  of  the  vulva,  and 
Nisot  and  Bumm  have  reported  cases  of  puerperal  diphtheria 
from  which  the  bacilli  were  cultivated.  It  is  in  nearly 
all  cases  a  purely  local  infection,  depending  upon  the 
presence  and  development  of  the  bacilli  upon  the  diseased 
mucous  membrane,  but  is  accompanied  by  a  serious  intoxi- 
cation resulting  from  the  absorption  from  the  local  lesions 
of  a  poisonous  metabolic  product  of  the  bacilli.  The  bacilli 
are  found  only  in  the  membranous  exudation,  and  are 
most  plentiful  in  its  older  portions. 

The  entrance  of  the  diphtheria  bacillus  into  the  internal 
organs  can  scarcely  be  regarded  as  a  frequent  occurrence, 
though  metastatic  occurrence  of  the  organism  with  and 
without  associated  staphylococci  and  streptococci,  and 
with  and  without  purulent  inflammations  have  from  time 
to  time  been  reported.  Such  a  case  of  septic  invasion  by 
the  diphtheria  bacillus,  with  a  synopsis  of  the  literature  to 
date,  is  given  by  Ucke.  f 

The  disease  pursues  a  course  of  variable  length,  in  favor- 
able cases  the  patient  recovering  gradually,  the  pseudo- 
membrane  first  disappearing,  leaving  an  inflamed  mucous 
membrane  behind  it,  upon  which  virulent  diphtheria  bacilli 
persist,  always  for  weeks  and  sometimes  for  months.  Smith  J 
describes  the  bacteriologic  condition  of  the  throat  in  diph- 
theria as  follows:  "The  microscope  informs  us  that  during 
the  earliest  local  manifestations  the  usual  scant  miscel- 
laneous bacterial  flora  of  the  mucosa  is  quite  suddenly 
replaced  by  a  rich  vegetation  of  the  easily  distinguishable 
diphtheria  bacillus.  Frequently  no  other  bacteria  are  found 

*  "  Amer.  Jour,  of  Obstet.  and  Dis.  of  Women  and  Children,"  Aug., 
1898. 

t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  original,  XLVI,  Heft  4,  March 
10,  1908,  p.  292. 

J  "Boston  Med.  and  Surg.  Jour.,"  1898,  I,  p.  157. 


Pathogenesis  4.63 

in  the  culture-tube.  This  vegetation  continues  for  a  few 
days,  then  gradually  gives  way  to  another  flora  of  cocci  and 
bacilli,  and  finally  the  normal  condition  is  reestablished." 

Diphtheria  bacilli  were  first  found  in  the  heart's  blood, 
liver,  spleen,  and  kidney,  by  Frosch.*  Kolisko  and  Pal- 
tauff  had  already  found  them  in  the  spleen,  and  other 
observers  in  various  lesions  of  the  deeper  tissues  and  occa- 
sionally in  the  organs.  In  the  blood  and  organs  it  is  com- 
monly associated  with  Streptococcus  pyogenes  and  some- 
times with  other  bacteria.  While  present  in  nearly  all  of 
the  inflammatory  sequelae  of  diphtheria,  the  Klebs-Loffler 
bacillus  probably  has  very  little  influence  in  producing  them, 
the  conditions  being  almost  invariably  associated  with  the 
pyogenic  cocci,  either  the  streptococci  or  staphylococci. 

Howard}  studied  a  case  of  ulcerative  endocarditis  caused 
by  the  diphtheria  bacillus,  and  Pearce  §  has  observed 
it  in  i  case  of  malignant  endocarditis,  19  out  of  24  cases  of 
broncho-pneumonia,  i  case  of  empyema,  16  cases  of  middle- 
ear  disease,  8  cases  of  inflammation  of  the  antrum  of  High- 
more,  i  case  of  inflammation  of  the  sphenoidal  sinuses,  i 
case  of  thrombosis  of  the  lateral  sinuses,  2  cases  of  abscesses 
of  the  cervical  glands,  and  in  esophagitis,  gastritis,  vulvo- 
vaginitis,  dermatitis,  and  conjunctivitis  following  or  asso- 
ciated with  diphtheria. 

In  animals  artificially  inoculated  with  the  diphtheria 
bacillus  the  resulting  lesions  resemble  those  seen  in  the 
human  subject,  in  that  they  consist  of  a  local  infection 
with  a  general  toxemia. 

Human  beings,  horses,  rabbits,  guinea-pigs,  mice,  kittens, 
and  young  pups  are  susceptible ;  rats  are  immune.  When  half 
a  cubic  centimeter  of  a  twenty-four-hour-old  bouillon  culture 
is  injected  beneath  the  skin  of  a  susceptible  animal,  the  bacilli 
multiply  at  the  point  of  inoculation,  producing  a  fibrinous  in- 
flammation with  edema.  The  animal  dies  about  the  third 
day.  When  examined  post-mortem  the  liver  is  found  en- 
larged and  sometimes  shows  minute  whitish  points,  which 
upon  microscopic  examination  prove  to  be  necrotic  areas  in 
which  the  cells  are  completely  degenerated,  and  the  chrom- 
atin  of  their  nuclei  scattered  about  in  granular  form.  Similar 

*  "Zeitschrift  fur  Hygiene,"  etc.,  1893,  XIII»  Heft  i. 

t  "Wiener  klin.  Wochenschrift,"  1889. 

i  "Amer.  Jour.  Med.  Sci.,"  Dec.,  1894. 

§  "Jour.  Boston  Soc.  of  Med.  Sci.,"  March,  1898. 


464  Diphtheria 

necrotic  foci,  to  which  attention  was  first  called  by  Oertel, 
are  present  in  nearly  all  the  organs  in  cases  of  death  from 
diphtheria  intoxication.  No  bacilli  are  present  in  these 
lesions.  Welch  and  Flexner  *  have  shown  these  foci  to 
be  common  to  numerous  intoxications,  and  not  peculiar  to 
diphtheria. 

The  lymphatic  glands  are  usually  enlarged,  and  the  adre- 
nals enlarged  and  hemorrhagic.  The  kidneys  show  paren- 
chymatous  degeneration.  There  is  no  inflammation  of  the 
fauces. 

Roux  and  Yersin  found  that  when  the  bacilli  were  intro- 
duced into  the  trachea  of  animals  opened  by  operation,  a 
typical  pseudo-membrane  was  formed,  and  that  diphtheritic 
palsy  sometimes  followed. 

Associated  Bacteria. — Streptococcus  pyogenes  and  Staphy- 
lococci  pyogenes  aureus  and  albus  are,  in  many  cases, 
found  in  association  with  the  diphtheria  bacillus,  especially 
when  severe  lesions  of  the  throat  exist. 

In  a  series  of  234  cases  carefully  and  statistically  studied 
by  Blasi  and  Russo-Travali,f  it  was  found  that  in  26  cases 
of  pseudo-membranous  angina  due  to  streptococci,  staphy- 
lococci,  colon  bacilli,  and  prieumococci,  2  patients  died, 
the  mortality  being  3.84  per  cent.  In  102  cases  of  pure 
diphtheria,  28  died,  a  mortality  of  27.45  per  cent.  Seventy- 
six  cases  showed  diphtheria  bacilli  and  staphylococci;  of 
these,  25,  or  32.89  per  cent.,  died.  Twenty  cases  showed 
the  diphtheria  bacilli  and  Streptococcus  pyogenes,  with  6 
deaths — 30  per  cent.  In  7  cases,  of  which  3,  or  43  per 
cent.,  were  fatal,  the  diphtheria  bacillus  was  in  combination 
with  streptococci  and  pneumococci.  The  most  dangerous 
forms  met  were  3  cases,  all  fatal,  in  which  the  diphtheria 
bacillus  was  found  in  combination  with  Bacillus  coli. 

In  157  cases  of  diphtheria  and  scarlatina  studied  at  the 
Boston  City  Hospital  by  Pearce,{  there  were  94  cases  of 
diphtheria,  46  cases  of  complicated  diphtheria  (29  with 
scarlet  fever,  n  with  measles,  and  5  with  measles  and 
scarlet  fever),  and  17  cases  of  scarlet  fever  (in  3  of  which 
measles  was  also  present). 

Of  the  94  cases  of  uncomplicated  diphtheria,  the  Klebs- 
Loffler  bacilli  were  present  in  the  heart's  blood  in  4,  twice 

*  "  Bull,  of  the  Johns  Hopkins  Hospital,"  Aug.,  1901. 

f  "Ann.  del'Inst.  Pasteur,"  1896,  p.  387. 

%  "Jour.  Boston  Soc.  of  Med  Sci.,"  March,  1898. 


Pathogenesis  465 

alone  and  twice  with  streptococci.  In  9  cases  the  strepto- 
coccus occurred  alone ;  in  i  case  the  pneumococcus  occurred 
alone.  In  the  liver  the  bacillus  was  found  in  24  cases, 
alone  in  1 2  and  together  with  the  streptococcus  in  12;  the 
streptococcus  occurred  in  27  cases,  alone  in  14,  with  the 
Klebs-Loffler  bacillus  in  12,  and  with  Staphylococcus 
pyogenes  aureus  in  i.  Staphylococcus  pyogenes  aureus 
occurred  in  4  cases,  alone  in  3  and  associated  with  the 
streptococcus  in  i.  The  pneumococcus  occurred  alone  in 
i  case. 

In  the  spleen  the  Klebs-Loffler  bacillus  occurred  eighteen 
times,  fifteen  times  alone  and  three  times  associated  with 
the  streptococcus.  The  streptococcus  occurred  in  24  cases, 
alone  in  21,  associated  with  the  Klebs-Loffler  bacillus  twice, 
and  with  Staphylococcus  pyogenes  aureus  once.  Staphy- 
lococcus pyogenes  occurred  twice,  once  alone  and  once  with 
the  streptococcus.  The  pneumococcus  occurred  twice  alone. 

In  the  kidney  the  Klebs-Loffler  bacillus  occurred  in  23 
cases,  in  15  alone,  in  5  associated  with  the  streptococcus, 
and  in  2  with  Staphylococcus  pyogenes  aureus.  The 
streptococcus  occurred  in  26  cases,  in  19  of  which  it  was  the 
only  organism  present.  Staphylococcus  pyogenes  aureus 
occurred  in  8  cases,  in  4  of  which  it  was  in  pure  culture. 
The  pneumococcus  occurred  four  times,  three  times  in  pure 
culture  and  once  with  the  Klebs-Loffler  bacillus. 

In  the  46  cases  of  complicated  diphtheria,  the  heart's 
blood  showed  pure  cultures  of  the  streptococcus  nine  times 
and  the  streptococcus  associated  with  the  Klebs-Loffler 
bacillus  once.  The  diphtheria  bacillus  occurred  alone  once. 

In  the  liver,  in  10  cases  streptococcus  occurred  alone,  in 
7  cases  associated  with  the  Klebs-Loffler  bacillus,  and  in  3 
cases  with  Staphylococcus  pyogenes  aureus.  The  diphtheria 
bacillus  occurred  in  pure  culture  in  5  cases. 

The  spleen  contained  streptococci  only  thirteen  times  and 
mixed  with  the  diphtheria  bacillus  twice.  The  diphtheria 
bacillus  was  found  in  pure  culture  in  5  cases. 

The  kidney  contained  pure  cultures  of  streptococci  in 
10  cases,  streptococci  associated  with  diphtheria  bacilli  five 
times,  and  with  Staphylococcus  pyogenes  aureus  three 
times.  The  diphtheria  bacillus  occurred  alone  in  7  cases. 
Staphylococcus  pyogenes  aureus  and  the  pneumococcus 
each  alone  once  and  both  together  once. 

"The  clinical  significance  of  this  general  infection  with  the 
30 


466  Diphtheria 

Klebs-Loffler  bacillus  is  not  apparent.  It  occurred  gener- 
ally, but  not  always,  in  the  gravest  cases,  or  those  known  as 
'septic'  cases.  It  is  probable  that  it  may  be  due  to  a 
diminished  resistance  to  the  tissue-cells,  or  of  the  germicidal 
power  of  the  blood.  In  this  series  of  fatal  cases  the  number 
of  infections  with  the  streptococcus  and  with  the  Klebs- 
Loffler  bacillus  was  about  even,  though  slightly  in  favor  of 
the  streptococcus." 

The  mixed  infections  add  to  the  clinical  diphtheria  the 
pathogenic  effects  of  the  associated  bacteria.  The  diphtheria 
bacillus  probably  begins  the  process,  growing  upon  the 
mucous  membrane,  devitalizing  it  by  its  toxin,  and  pro- 
ducing coagulation-necrosis.  Whatever  pyogenic  germs 
happen  to  be  present  are  thus  afforded  an  opportunity  to 
enter  the  tissues  and  add  suppuration,  gangrene,  and  re- 
mote metastatic  lesions  to  the  already  existing  ulceration. 

Diphtheritic  inflammations  of  the  throat  are  not  always 
accompanied  by  the  formation  of  the  pseudo-membrane, 
but  in  some  cases  a  rapid  inflammatory  edema  in  the  larynx, 
without  a  fibrinous  surface  coating,  may  cause  fatal  suffoca- 
tion, only  a  bacteriologic  examination  revealing  the  true 
nature  of  the  disease. 

Lesions. — The  pseudo-membrane  characterizing  diph- 
theria consists  of  a  combined  necrosis  of  the  tissues  acted 
upon  by  the  toxin  and  coagulation  of  an  inflammatory 
exudate.  When  examined  histologically  it  is  found  that 
the  surface  of  the  mucous  membrane  is  chiefly  affected. 
The  superficial  layers  of  cells  being  embedded  in  coagulated 
exudate- — fibrin — and  show  a  peculiar  hyaline  degeneration. 
Sometimes  the  membrane  seems  to  consist  exclusively  of 
hyaline  cells ;  sometimes  the  fibrin  formation  is  secondary  to 
or  subsequent  to  the  hyaline  degeneration.  Leukocytes 
caught  in  the  fibrin  also  become  hyaline.  From  the  super- 
ficial layer  the  process  may  descend  to  the  deepest  layers, 
all  of  the  cells  being  included  in  the  coagulated  fibrin  and 
showing  more  or  less  hyaline  degeneration.  The  walls  of  the 
neighboring  capillaries  also  become  hyaline,  and  the  necrotic 
mass  forms  the  diphtheritic  membrane.  The  laminated 
appearance  of  the  membrane  probably  depends  upon  the 
varying  depths  affected  at  different  periods,  or  upon  differ- 
ences in  the  process  by  which  it  has  been  formed.  The 
pseudo-membrane  is  continuous  with  the  subjacent  tissues 
by  a  fibrinous  reticulum,  and  is  in  consequence  removed 


Specificity  467 

with  difficulty,  leaving  an  abraded  surface.  When  the  mem- 
brane is  divulsed  during  the  course  of  the  disease,  it  imme- 
diately forms  anew  by  the  coagulation  of  the  inflammatory 
exudate. 

The  coagulation -necrosis  seems  to  depend  upon  the  local 
effect  of  the  toxin.  Morax  and  Elmassian  *  found  that 
when  strong  diphtheria  toxin  is  applied  to  the  conjunctiva 
of  rabbits  every  three  minutes  for  eight  or  ten  hours, 
typical  diphtheritic  changes  are  produced. 

Flexnerf  has  made  a  study  of  the  minute  lesions  caused 
by  bacterial  toxins  and  especially  of  the  diphtheria  toxin, 
and  Councilman,  Mallory,  and  Pearce,J  of  both  gross  and 
minute  lesions,  that  the  thorough  student  should  read. 

Specificity. — Herman  Biggs,  §  in  an  interesting  discus- 
sion of  the  occurrence  of  the  diphtheria  bacillus  and  its 
relation  to  diphtheria,  comes  to  the  following  conclusions: 

1.  "When  the  diphtheria  bacillus  is  found   in  healthy 
throats,  investigation  almost  always  shows  that  the  indi- 
viduals have   been   in   contact   with   cases   of   diphtheria. 
The  presence  of  the  bacillus  in  the  throat,   without  any 
lesion,  does  not,  of  course,  indicate  the  existence  of  the 
disease." 

2.  "The    simple   anginas   in    which   virulent   dipntheria 
bacilli  are  found  are  to  be  regarded  from  a  sanitary  stand- 
point in  exactly  the  same  way  as  the  cases  of  true  diph- 
theria." 

3.  "Cases   of   diphtheria    present   the   ordinary   clinical 
features  of  diphtheria,  and  show  the  Klebs-Loffler  bacilli." 

4.  "Cases  of  angina  associated  with  the  production  of 
membrane  in  which  no  diphtheria  bacilli  are  found  might 
be  regarded  from  a  clinical  standpoint  as  diphtheria,  but 
bacteriological  examination  shows  that  some  other  organ- 
ism than  the  Klebs-Loffler  bacillus  is  the  cause  of  the  pro- 
cess." 

Any  skepticism  of  the  specificity  of  the  diphtheria  bacillus 
on  my  own  part  was  dispelled  by  a  somewhat  unique  ex- 
perience. Without  having  been  previously  exposed  to 
diphtheria  while  experimenting  in  the  laboratory  I  acciden- 

*  "Ann.  de  1'Inst.  Pasteur,"  1898,  p.  210. 
f"  Johns  Hopkins  Hospital  Reports,"  vi,  259. 
J "  Diphtheria  :   A  Study  of  the  Bacteriology  and  Pathology  of 
Two  Hundred  and  Twenty  Fatal  Cases,"  1901. 

§  "Amer.  Jour.  Med.  Sci.,"  Oct.,  1896,  vol.  xxn,  No.  4,  p.  411. 


468  Diphtheria 

tally  drew  a  living  virulent  culture  of  the  diphtheria  bacillus 
through  a  pipet  into  my  mouth.  Through  carelessness  no 
precautions  were  taken  to  prevent  serious  consequences,  and 
two  days  later  my  throat  was  filled  with  typical  pseudo- 
membrane  which  private  and  Health  Board  bacteriologic 
examinations  showed  to  contain  pure  cultures  of  the  Klebs- 
Loffler  bacilli. 

Some  have  been  led  to  doubt  the  specificity  of  the  diphtheria 
bacillus  because  of  the  existence  of  what  is  called  the  pseudo- 
diphtheria  bacillus  or  bacillus  of  Hofmann  (q.  v.).  Bomstein* 
found  that  though  it  was  possible  to  modify  the  activity  of 
virulent  bacilli,  and  bring  back  the  virulence  of  non- virulent 
diphtheria  bacilli,  it  was  impossible  to  make  the  pseudo- 
diphtheria  bacillus  virulent.  Denny  f  also  found  that  the 
morphology  of  the  two  organisms  was  continually  different 
when  they  were  grown  upon  the  same  medium  for  the  same 
length  of  time,  and  that  the  short  pseudodiphtheria  bacillus 
never  showed  any  tendency  to  develop  into  the  large  clubbed 
forms  characteristic  of  the  true  diphtheria  organism.  The 
chief  points  of  difference  between  the  bacilli  are  that  the 
pseudodiphtheria  bacillus,  when  grown  upon  blood-serum,  is 
short  and  stains  uniformly;  that  cultures  grown  in  bouillon 
develop  more  rapidly  at  a  temperature  of  from  20°  to  22°  C. 
than  those  of  the  true  bacillus;  and  that  the  pseudobacillus 
is  not  pathogenic  for  animals. 

Contagion. — The  diphtheria  bacilli,  being  always  present 
in  the  throats  of  patients  suffering  from  diphtheria,  con- 
stitute the  element  of  contagion,  and  by  being  accidentally 
discharged  from  the  nose  and  mouth  during  coughing, 
sneezing,  vomiting,  etc.,  endanger  whoever  comes  in  contact 
with  the  patient. 

The  results  obtained  by  Biggs,  Park,  and  Beebe  in  New 
York  are  of  great  interest.  Bacteriologic  examinations 
conducted  in  connection  with  the  Health  Department  of 
New  York  city  show  that  virulent  diphtheria  bacilli  may 
be  found  in  the  throats  of  convalescents  from  diphtheria, 
as  long  as  five  weeks  after  the  discharge  of  the  membrane 
and  the  commencement  of  recovery,  and  that  they  exist 
not  only  in  the  throats  of  the  patients  themselves,  but  also 
in  those  of  their  caretakers,  who,  while  not  themselves 
infected,  may  be  the  means  of  conveying  the  disease  germs 
*  "Archiv  Russes  de  Path.,"  etc.,  Aug.  31,  1902. 
t  American  Public  Health  Association,  1902. 


Specificity  469 

from  the  sick-room  to  the  outer  world.  Still  more  extra- 
ordinary are  the  observations  of  Hewlett  and  Nolen,*  that 
the  bacilli  remained  in  the  throats  of  patients  seven, 
nine,  and  in  one  case  twenty-three  weeks  after  convalescence. 
The  hygienic  importance  of  this  observation  must  be  ap- 
parent to  all  readers,  and  serves  as  further  evidence  why 
thorough  isolation  should  be  practised  in  connection  with  the 
disease. 

Neumann  f  found  that  virulent  diphtheria  bacilli  may 
occur  in  the  nose  with  the  production  of  what  seems  to  be 
a  simple  rhinitis  as  well  as  a  pseudo-membranous  rhinitis. 


Fig.  152.  —  Wesbrook's  types  of  Bacillus  diphtheriae:  a,  c,  d,  Granular 
types;  a1,  c1,  d1,  barred  types;  a2,  c2,  d2,  solid  types.     X  1500. 


Such  cases,  not  being  segregated,  may  easily  serve  to  spread 
the  contagion  of  the  disease. 

Wesbrook,  and  Wilson  and  McDaniel  J  have  found  it  con- 
venient to  describe  three  chief  types  of  the  diphtheria 
bacillus  as  it  occurs  in  twenty-four-hour-old  cultures  on 
Loffler's  blood-serum,  sent  to  the  laboratory  for  diagnosis. 
The  classification  places  all  types  in  three  general  groups: 
(a)  granular,  (6)  barred,  and  (c)  solid  or  evenly  staining 
forms.  Each  group  is  subdivided  into  types  based  on  the 

*"Brit.  Med.  Jour.,"  Feb.  i,  1896. 

t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Jan.  24,  1902,  Bd.  xxxi, 
No.  2,  p.  41. 

J  "Trans.  Assoc.  Amer.  Phys.,"  1900. 


47°  Diphtheria 

shape  and  size  of  the  bacilli.  A  study  of  variations  in  the 
sequence  of  types  in  series  of  cultures  derived  from  clinical 
cases  of  diphtheria  shows  that  (a)  granular  types  are  usually 
the  most  predominant  forms  at  the  outset  of  the  disease; 
(6)  the  granular  types  usually  give  place  wholly  or  in  part 
to  barred  and  solid  types  shortly  before  the  disappearance 
of  diphtheria-like  organisms;  (c)  solid  types,  by  many 
observers  called  "pseudo-diphtheria  bacilli,"  may  cause 
severe  clinical  diphtheria.  Solid  types  may  sometimes  be 
replaced  by  granular  types  when  convalescence  is  established 
and  just  before  the  throat  is  cleared  of  diphtheria-like  bacilli. 

From  these  data  the  writers  conclude  that  it  is  not  safe 
to  base  an  opinion  regarding  the  maintenance  of  quarantine 
upon  the  bacterioscopic  findings  independently  of  the  clinical 
history  of  the  case. 

The  occurrence  of  true  diphtheria  bacilli  in  the  throats 
of  healthy  persons  has  been  a  stumbling-block  to  many 
practitioners  uninformed  upon  bacteriologic  subjects,  who 
fail  to  account  for  its  presence  and  also  fail  to  realize  how 
rare  its  appearance  under  such  circumstances  really  is. 

Park  *  found  virulent  diphtheria  bacilli  in  about  i  per 
cent,  of  the  healthy  throats  examined  in  New  York  city, 
but  diphtheria  was  prevalent  in  the  city  at  the  time,  and 
no  doubt  most  of  the  persons  in  whose  throats  they  existed 
had  been  in  contact  with  cases  of  diphtheria.  He  very 
properly  concludes  that  the  members  of  a  household  in 
which  a  case  of  diphtheria  exists,  though  they  have  not 
the  disease,  should  be  regarded  as  possible  sources  of 
danger,  until  cultures  made  from  their  throats  show  that 
the  bacilli  have  disappeared. 

In  connection  with  the  contagiousness  of  diphtheria  the 
recent  experiments  of  Reyes  are  interesting.  He  has 
demonstrated  that  in  absolutely  dry  air  diphtheria  bacilli 
die  in  a  few  hours.  Under  ordinary  conditions  their  vitality, 
when  dried  on  paper,  silk,  etc.,  continues  for  but  a  few  days, 
though  sometimes  they  can  live  for  several  weeks.  In  sand 
exposed  to  a  dry  atmosphere  the  bacilli  die  in  five  days 
in  the  light ;  in  sixteen  to  eighteen  days  in  the  dark.  When 
the  sand  is  exposed  to  a  moist  atmosphere,  the  duration 
of  their  vitality  is  doubled.  In  fine  earth  they  remained 

*  "Report  on  Bacteriological  Investigations  and  Diagnosis  of  Diph- 
theria, from  May  4,  1893,  to  May  4,  1894,"  "Scientific  Bulletin  No.  i," 
Health  Department,  city  of  New  York. 


Diphtheria  Antitoxin  471 

alive  seventy-five  to  one  hundred  and  five  days  in  dry  air, 
and  one  hundred  and  twenty  days  in  moist  air. 

Diphtheria  Antitoxin. — Behring  *  discovered  that  the 
blood  of  animals  rendered  immune  against  diphtheria  by 
inoculation,  first  with  attenuated  and  then  with  virulent 
organisms,  contained  a  neutralizing  substance  (Anti-korper] 
capable  of  annulling  the  effects  of  the  bacilli  or  the  toxin  when 
simultaneously  or  subsequently  inoculated  into  susceptible 
animals.  This  substance,  held  in  solution  in  the  blood- 
serum  of  the  immunized  animals,  is  the  diphtheria  antitoxin. 
For  method  of  preparing  the  antitoxins  see  Antitoxins. 
The  serum  may  be  employed  for  purposes  of  prophylaxis  or 
for  treatment. 

Prophylaxis. — The  serum  can  be  relied  upon  for  prophy- 
laxis in  cases  of  exposure  to  diphtheria  infection.  In  most 
cases  a  single  dose  of  500  units  is  sufficient  for  the  purpose. 
The  protection  thus  afforded  does  not  continue  longer  than 
about  three  months.  The  transitory  nature  of  the  immun- 
ity afforded  by  prophylactic  injections  of  antitoxin  is  prob- 
ably dependent  upon  the  fact  that  the  antitoxin  is  slowly 
excreted  through  the  kidneys. 

Treatment. — Diphtheria  antitoxin  is  always  to  be  admin- 
istered by  the  hypodermic  method  at  some  point  where  the 
skin  is  loose.  Some  clinicians  prefer  to  inject  into  the  ab- 
dominal wall;  some,  into  the  tissues  of  the  back.  A  slightly 
painful  swelling  is  formed,  which  usually  disappears  in  a  short 
time.  In  a  few  cases  sudden  death,  with  symptoms  sug- 
gesting anaphylaxis  (q.  v.),  has  followed  the  injection. 

Ehrlich  asserts  that  a  dose  of  500  units  is  valueless  for  the 
treatment  of  diphtheria,  2000  units  being  probably  an  aver- 
age dose  for  an  adult  and  1000  units  for  a  child.  As  the 
remedy  is  practically  harmless,  it  is  far  better  to  err  on  the 
side  of  administering  too  much  than  on  that  of  not  enough. 
Forty  thousand  units  have  been  administered  to  a  moribund 
child  with  resulting  cure.  The  administration  of  the 
remedy  should  be  repeated  in  twelve  hours  if  the  disease 
is  one  or  two  days  old,  in  six  hours  if  three  or  four  days  old, 
in  four  hours  if  still  older.  The  serum  may  have  to  be  given 
two,  three,  four,  or  even  more  times,  according  to  the  case. 
Occasionally  there  is  an  outbreak  of  local  urticaria — rarely 
general  urticaria.  Sometimes  considerable  local  erythema 

*"  Deutsche  med.  Wochenschrift,"  1890,  Nos.  49  and  50;  "Zeit- 
schrift  fiir  Hygiene,"  xn,  i,  1892. 


472  Diphtheria 

results.  Fever  and  pain  in  the  joints  (serum  disease  of  von 
Pirquet)  also  occur,  especially  if  the  patients  have  been  pre- 
viously treated  with  horse-serum.  Serums  of  high  unit 
strength  can  be  given  with  the  ordinary  hypodermic  syringe ; 
those  of  lower  strength,  of  which  a  larger  quantity  is  required, 
must  be  given  with  a  special  "antitoxin  syringe."  The  syr- 
inge should  always  be  carefully  sterilized  by  boiling,  and  the 
packings,  etc.,  found  to  be  in  good  condition  before  it  is  filled 
with  antitoxin. 

Diphtheria  paralysis  is  said  to  be  more  frequent  after  the 
use  of  antitoxin  than  in  cases  treated  without  it.  In  a  paper 
upon  this  subject  I*  have  shown  that  this  is  to  be  expected, 
as  the  palsies  usually  occur  after  bad  cases  of  the  disease, 
of  which  a  far  greater  number  recover  when  antitoxin  is  used 
for  treatment.  The  subject  has  been  worked  over  in  an  in- 
teresting manner,  from  the  experimental  side,  by  Rosenau.  f 

An  interesting  collection  of  statistics  upon  the  antitoxic 
treatment  of  diphtheria  in  the  hospitals  of  the  world  has 
been  published  by  Professor  Welch,  t  who,  excluding  every 
possible  error  in  the  calculations,  "shows  an  apparent  re- 
duction of  case-mortality  of  55.8  per  cent." 

Nothing  should  so  impress  the  clinician  as  the  necessity 
of  beginning  the  antitoxin  treatment  early  in  the  disease. 
Welch's  statistics  show  that  1115  cases  of  diphtheria  treated 
in  the  first  three  days  of  the  disease  yielded  a  fatality  of 
8.5  per  cent.,  whereas  546  cases  in  which  the  antitoxin 
was  first  injected  after  the  third  day  of  the  disease  yielded 
a  fatality  of  27.8  per  cent. 

On  the  other  hand,  it  can  scarcely  be  said  that  any  time 
is  too  late  to  begin  the  serum  treatment,  for  the  experiences 
of  Burroughs  and  McCollum  in  the  Boston  City  Hospital 
show  that  by  immediate  and  repeated  administration  of 
very  large  doses  of  the  serum,  apparently  hopeless  cases 
far  advanced  in  the  disease,  may  often  be  saved. 

After  the  toxin  has  occasioned  destructive  organic  lesions 
of  the  nervous  system  and  in  the  various  organs  and  tissues 
of  the  body,  no  amount  of  neutralization  can  restore  the 
integrity  of  the  parts,  and  in  such  cases  antitoxin  must 
fail. 

*  "Medical  Record,"  New  York,  1897. 

t  "Bulletin  No.  38  of  the  Hygienic  Laboratory,  U.  S.  Public  Health 
and  Marine  Hospital  Service,"  Washington,  D.  C.,  1907. 

t  "Bull,  of  the  Johns  Hopkins  Hospital,"  July  and  Aug.,  1895. 


Diphtheria  Antitoxin  473 

The  occurrence  of  tetanus  following  the  employment  of 
serum  drawn  from  a  horse  suffering  from  tetanus  was  ob- 
served in  a  number  of  cases  in  St.  Louis.  In  rare  cases 
in  which  local  and  metastatic  abscesses  have  been  observed, 
the  condition  is  probably  correctly  attributable  to  infection 
from  the  patient's  skin  or  from  the  syringe. 

I  have  found  that  the  serums  are  by  no  means  regular 
in  the  rapidity  of  deterioration,  so  that  no  very  old  serum 
should  be  used. 

Freezing  is  without  effect  upon  the  serum  and  ordinary 
temperature  changes  are  harmless  to  it.  The  antitoxic 
power  is  destroyed  at  60°  C.,  the  point  at  which  the  serum 
coagulates.  The  antitoxin  is  precipitated  with  the  globulins.* 

The  serums  from  different  horses  probably  vary  much 
in  both  their  irritant  and  globulicidal  .properties,  so  that 
mixed  serums  from  a  number  of  horses  may  be  preferable  to 
that  from  a  single  horse. 

A  very  interesting  paper  by  Parkf  shows  the  effect  of  the 
introduction  of  antitoxin  upon  the  death-rate  from  diphtheria 
and  the  advantages  of  its  employment.  From  it  the  follow- 
ing table  is  taken: 

"  Combined  statistics  of  deaths  and  death-rates  from  diphtheria  and 
croup  in  New  York,  Brooklyn,  Boston,  Pittsburgh,  Philadelphia,  Berlin, 
Cologne,  Breslau,  Dresden,  Hamburg,  Konigsberg,  Munich,  Vienna, 
Soudan,  Liverpool,  Glasgow,  Paris,  and  Frankfort: 

Year.  population.        Deathsjromdiphtheria    Deaths,per 

1890 .16,526,135  H,059  66.9 

1891 17,689,146  12,389  70 

1892 18,330,787  14,200  77.5 

1893 18,467,970  15,726  80.4 

1894 19.033,902  15,125  79-9 

i«95J 19,143,188  10,657  55.6 

1896 19,489,682  9,651  49.5 

1897 19,800,629  8,942  45.2 

1898 20,037,918  7,170  35.7 

1899 20,358,837  7,256  35.6 

1900 20,764,614  6,791  32.7 

1901 20,874,572  6,104  29.2 

1902 21,552,398  5,630  26.1 

1903 .21,865,299  5,117  23.4 

1904 22,532,848  4,917  21.8 

1905 22,790,000  4,323  19.0 

*  See  paper  by  J.  P.  Atkinson,  ''Journal  of  Experimental  Medicine," 
Sept.  and  Nov.,  1899,  vol.  iv,  Nos.  5  and  6. 

t"  Journal  of  the  Amer.  Med.  Assoc.,"  Feb.  17,  1912,  LVIII,  No.  7, 
P-  453- 

J  Introduction  of  antitoxin  treatment. 


474 


Diphtheria 


BACILLI  RESEMBLING  THE  DIPHTHERIA  BACILLUS. 
BACILLUS  HOFMANNI. 

The  pseudodiphtheria  bacillus  (bacillus  of  Hofmann- 
Wellenhof*) — Bacillus  pseudodiphthericus — was  first  found 
by  L/offlerf  in  diphtheria  pseudomembranes  and  in  the 
healthy  mouth  and  pharynx.  Ohlmacher  has  found  it  with 
other  bacteria  in  pneumonia ;  Babes,  in  gangrene  of  the  lung ; 
and  Howard,  t  in  a  case  of  ulcerative  endocarditis  not  suc- 
ceeding diphtheria. 


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Fig-  153- — Pseudodiphtheria  bacilli. 

Park§  found  that  all  bacilli  with  the  typical  morphology 
of  the  diphtheria  bacillus,  found  in  the  human  throat,  are 
virulent  Klebs-L/ofrler  bacilli,  while  forms  found  in  the  throat 
closely  resembling  them,  but  more  uniform  in  size  and  shape, 
shorter  in  length,  and  of  more  homogeneous  staining  proper- 
ties with  Loffler's  alkaline  methylene-blue  solution,  can  with 
reasonable  safety  be  regarded  as  pseudodiphtheria  bacilli, 
especially  if  it  be  found  that  they  produce  an  alkaline  rather 
than  an  acid  reaction  by  their  growth  in  bouillon.  The 
pseudodiphtheria  bacilli  were  found  in  about  i  per  cent,  of 

*  "Wiener  klin.  Woch.,"  1888,  No.  3. 
t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  n,  105. 
t  "Bull,  of  the  Johns  Hopkins  Hospital,"  1893,  30. 
§  "Scientific  Bulletin  No.    i,"   Health   Department,   city  of    New 
York,  1895. 


Bacilli  Resembling  the  Diphtheria  Bacillus     475 

throats  examined  in  New  York;  they  seem  to  have  no  rela- 
tionship to  diphtheria,  and  are  never  virulent. 

Morphology. — This  micro-organism  bears  a  more  or  less 
marked  resemblance  to  Bacillus  diphtherias,  but  differs  in 
certain  particulars  that  usually  make  it  possible  to  recognize 
and  identify  it.  It  is  shorter  and  stouter  than  its  relative, 
is  straight,  usually  slightly  clubbed.  It  usually  stains  in- 
tensely, and  commonly  shows  but  one  unstained  transverse 
band.  When  the  bacilli  are  short  and  have  a  single  band, 
they  may  resemble  cocci.  When  longer  they  may  show  two 
transverse  bands. 

There  are  no  flagella  and  no  spores. 

Staining. — The  organism  stains  intensely  and  more  uni- 
formly than  Bacillus  diphtherias .  When  colored  by  Neisser's 
or  Roux's  method,  no  metachromatic  end  bodies  can  be 
defined. 

Cultivation. — The  organism  is  usually  discovered  in 
smears  made  for  the  diagnosis  of  diphtheria,  and  sometimes 
occasions  considerable  confusion  through  its  cultural  simi- 
larities and  morphologic  resemblances  to  Bacillus  diphtherias. 
It  grows  more  luxuriantly  upon  the  ordinary  culture-media 
than  B.  diphtherias.  The  colonies  are  larger,  less  transpar- 
ent and  whiter,  as  seen  upon  agar-agar.  In  bouillon  there  is 
more  marked  clouding  and  less  marked  pellicle  formation. 
Upon  Loffler's  blood-serum  the  cultures  are  too  much  alike 
to  be  easily  differentiated. 

G.  F.  Petri  *  found  no  substances  in  filtrates  of  cultures  of 
Hofmann's  bacillus  capable  of  neutralizing  diphtheria  anti- 
toxin; he  also  found  that  horses  immunized  with  large 
quantities  of  filtrates  of  the  Hofmann  bacillus  did  not  pro- 
duce any  antitoxin  to  diphtheria  toxin.  Eleven  different 
cultures  were  studied  and  the  results  are  very  important. 

Cobbettf  and  Knapp|  show  that  there  is  a  chemicobio- 
logic  difference  between  the  true  and  pseudodiphtheria 
bacilli,  in  that  the  pseudobacillus  does  not  ferment  dextrin 
or  any  of  the  sugars  as  the  true  bacillus  does. 

Chemistry. — The  chemical  peculiarities  of  the  culture 
serve  to  make  certain  that  Bacillus  hofmanni  is  an  independ- 
ent micro-organism.  Under  no  circumstances  does  it  pro- 
duce or  can  it  be  made  to  produce  toxin.  Under  no  circum- 

*  "Jour,  of  Hygiene,"  vol.  v,  No.  2,  April,  1905,  p.  134. 
f  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1898,  xxm,  395. 
t  "Jour,  of  Med.  Research,"  xn  (N.  S.,  vol.  vn),  1904,  p.  475. 


476  Diphtheria 

stances  can  it  be  made  to  produce  acid  through  the  decompo- 
sition of  sugars. 

Pathogenesis. — Bacillus  hofmanni  is  not  pathogenic  for 
any  of  the  laboratory  animals.  Animals  immunized  to 
repeated  injections  of  its  cultures  acquire  no  resistance  to 
Bacillus  diphtheriae. 

Dr.  Alice  Hamilton*  carefully  studied  29  organisms,  of 
which  26  corresponded  fully  with  the  pseudodiphtheria 
bacilli.  They  were  divisible  into  three  groups:  I,  Those 
non-pathogenic  for  guinea-pigs;  II,  those  that  produce 
general  bacteremia  in  guinea-pigs,  and  are  neutralized  by 
treatment  with  the  serum  of  a  rabbit  immunized  against  a 
member  of  the  group;  III,  organisms  which  form  gas  in 
glucose  media,  produce  bacteremia  in  guinea-pigs,  and  are 
neutralized  neither  by  diphtheria  nor  by  pseudodiphtheria 
antitoxin.  Some  of  the  organisms  of  the  second  group  are 
also  pathogenic  for  man.  Instead  of  regarding  the  pseudo- 
diphtheria  bacillus  as  a  harmless  saprophyte,  Dr.  Hamilton 
believes  it  an  important  organism  explaining  some  of  the 
paradoxes  that  we  find  at  hand.  Thus,  cases  of  supposed 
diphtheria  irremediable  by  or  deleteriously  affected  by  anti- 
toxic serum  may  depend  upon  one  of  these  organisms.  It 
is  also  probably  one  of  them  that  Councilman  found  in  his 
case  of  "general  infection  by,  Bacillus  diphtheriae,"  and  that 
Howard  encountered  in  his  case  of  acute  ulcerative  endo- 
carditis without  diphtheria,  from  the  valves  of  whose  heart 
cultures  of  a  diphtheria-like  organism  not  pathogenic  for 
guinea-pigs  were  isolated. 

The  still  more  recent  and  comprehensive  work  of  Clark  | 
shows  that  no  kind  of  manipulation  is  capable  of  so  modifying 
Bacillus  hofmanni  as  to  make  its  identity  with  B.  diphtheriae 
in  the  least  likely.  Clark  is,  however,  willing  to  admit  the 
probability  that  the  organisms  may  have  descended  from  a 
common  stock. 

BACILLUS  XEROSIS. 

This  bacillus  was  first  described  in  1884  by  Kutschbert  and 
Neisser,t  who  regarded  it  as  the  cause  of  xerosis  conjunctivae, 
having  found  it  upon  the  conjunctiva  in  that  disease.  It  has, 
however,  been  so  frequently  found  upon  the  normal  conjunc- 

*  "Jour.  Infectious  Diseases,"  1904,  i,  p.  690. 

t  "Journal  of  Infectious  Diseases,"  vn,  1910,  335. 

t  "Deutsche  med.  Wochenschrift,"  1884,  Nos.  21,  24. 


Bacillus  Xerosis  477 

tiva  that  it  can  no  longer  be  looked  upon  as  pathogenic.  It 
is  also  found  upon  other  mucous  membranes  than  the  con- 
junctiva; thus,  Leber  found  it  in  the  mouth,  the  pelvis  of 
the  kidney,  and  in  intestinal  ulcers.  From  the  investigations 
of  Sattler,  Frankel  and  Franke,  Schleich,  Weeks,  Fick,  Baum- 
garten,  and  others  it  appears  that  Bacillus  xerosis  is  a  harm- 
less saprophyte  that  is  occasionally  found  upon  the  con- 
junctiva. Happening  to  be  found  in  xerosis  it  was  accorded 
undue  distinction. 

Morphology. — It  resembles  Bacillus  diphtheriae  very 
closely,  but  is  probably  a  little  shorter.  The  ends  are  clubbed, 
and  in  them  metachromatic  bodies  are  stained  by  Neisser's 
and  Roux's  methods. 

There  is  no  motility;  there  are  no  flagella  and  no  spores. 

Cultivation. — Upon  Loffler's  medium  and  other  media 
commonly  used  for  the  diagnosis  of  diphtheria,  the  organism 
grows  with  so  close  resemblance  to  the  Bacillus  diphtheriae  as 
to  make  the  differentiation  difficult.  Transplanted  to  other 
media,  it  continues  to  resemble  B.  diphtheriae. 

Chemistry. — The  organism  is  incapable  of  forming  any 
toxin.  It  ferments  sugars  like  Bacillus  diphtheriae,  with  the 
exception  of  saccharose,  which  B.  xerosis  ferments,  but  which 
B.  diphtheriae  cannot  ferment.  B.  xerosis  also  fails  to  fer- 
ment dextrin,  which  B.  diphtheria  ferments. 

These  sugar-decomposing  properties  form  the  most  reliable 
methods  of  differentiating  Bacillus  diphtheriae,  B.  hofmanni, 
and  B.  xerosis. 

Pathogenesis. — The  organism  is  not  pathogenic  for 
man  and  is  certainly  not  the  cause  of  xerosis.  It  is  not  toxi- 
cogenic  and  is  not  known  to  be  pathogenic  for  any  animal. 


CHAPTER   XII. 

VINCENT'S  ANGINA. 

VINCENT'S  angina  is  an  acute,  specific,  infectious,  pseudo- 
membranous  form  of  pharyngitis  or  tonsillitis  characterized 
by  the  formation  of  a  soft  yellowish -green  exudate  upon  the 
mucous  membranes,  which,  when  removed,  leaves  a  bleeding 
surface  which  remains  an  ulcer.  Sometimes  these  ulcers  are 
superficial,  sometimes  they  are  deep,  necrotic,  and  fetid. 
There  is  considerable  pain  on  swallowing,  some  fever,  and 
some  prostration.  The  patient  not  infrequently  keeps  up  and 
about,  though  feeling  very  badly.  The  ulcerations  sometimes 
persist  for  several  months.  As  there  is  considerable  swelling 
of  the  glands  of  the  neck  and  as  the  pseudomembrane  is 
sometimes  quite  distinct,  the  disease  is  apt  to  be  mistaken  for 
diphtheria,  and  may  be  differentiated  from  it  only  by  a  bacteri- 
ologic  examination.  When  such  an  examination  is  made  two 
apparently  different  micro-organisms  may  be  found.  The 
first  is  the  Bacillus  fusiformis;  the  second,  Spirochaeta  vin- 
centi. 

BACILLUS  FUSIFORMIS  (BABES  (?)). 

In  1882  Miller*  described  a  fusiform  bacillus  that  occurred 
in  small  numbers  between  the  gums  and  the  teeth  and  in 
cavities  in  carious  teeth  in  the  human  mouth.  In  1884 
Cornil  and  Babes f  also  described  a  fusiform  bacillus  which 
seems  to  be  somewhat  different,  that  occurred  in  a  ne- 
crotic exudation  from  a  pseudomembranous — diphtheritic 
— pharyngitis  in  school  children.  Lammershirt,  Vincent, 
Nicolle,  Plaut,  and  others  observed  similar  cases.  Later 
Lichtowitz  and  Sabrazes  observed  great  numbers  of  fusiform 
bacilli  in  the  pus  of  a  maxillary  empyema.  Elders  and  Matz- 
enauer  observed  similar  organisms  in  noma.  Fusiform 
bacilli  are,  therefore,  not  infrequently  associated  with  ne- 
crotic processes  of  various  kinds.  Similar  but  not  identical 
bacilli  were  found  by  Babes  in  the  gums  of  scorbutic  patients. 

*  "Micro-organisms  of  the  Human  Mouth." 
t  "Les  Bacteries,"  1884. 
478 


Relation  of  the  Organisms  to  One  Another     479 

SPIROCH^TA  VINCENTI  (PLAUT- VINCENT). 

Plaut*  and  Vincent  t  observe  that  in  the  ulcerative  and 
necrotic  pharyngitis  described,  together  with  the  fusiform 
bacilli,  there  were  varying  numbers  of  spiral  organisms. 
These  were  difficult  to  stain,  always  took  faint  but  uniform 
coloring,  varied  in  length,  and  showed  such  irregular  and 
non-uniform  undulations  as  to  appear  more  serpentine  than 
"corkscrew-like."  They  seem  never  to  occur  without  as- 
sociated fusiform  bacilli.  The  writers  believed  these  organ- 
isms and  not  the  bacilli  to  be  the  cause  of  the  angina,  but  the 
relation  of  the  organisms  to  one  another  and  to  the  morbid 
conditions  with  which  they  were  associated  was  a  point  long 
under  debate,  since  none  of  those  studying  either  organism 
succeeded  in  artificially  cultivating  them. 


RELATION  OF  THE  ORGANISMS  TO  ONE  ANOTHER. 

We  have,  in  Vincent's  angina,  to  do  with  two  micro-organ- 
isms that  occur  in  habitual  association.  Neither  was  found  to 
be  cultivable  by  the  earlier  writers.  The  spirochaeta  could 
not  be  cultivated  by  Vincent,  and  of  the  various  fusiform 
bacilli,  one  found  by  Babes  in  scurvy,  which  was  obviously 
different  from  the  others,  was  alone  susceptible  of  cultivation. 
Later,  however,  reports  were  made  of  the  growth  of  the  organ- 
isms in  mixed  cultures.  Still  later,  Veillon  and  Zuber, 
Ellermann,  Weaver,  and  Tunnicliff  were  able  to  secure  pure 
cultures  of  the  fusiform  bacillus.  Quite  a  number  of  writers 
reached  the  conclusion  that  the  organisms  were  not  different, 
but  were  different  stages  of  the  same  organism.  This  was 
finally  proved  by  Tunnicliff,  J  who  found  that  in  pure  cultures 
of  Bacillus  fusiformis,  after  forty-eight  hours,  spiral  organisms 
resembling  those  seen  in  smear  preparations  from  the  original 
source  were  found.  From  Tunnicliff 's  results  it  must  be 
concluded  that  Bacillus  fusiformis  and  Spirochaeta  vincenti 
are  .identical  organisms  in  different  stages  of  their  life-history. 
Which,  however,  is  the  perfect  form  is  not  known,  what  the 
true  nature  of  the  organism  is,  is  not  known,  nor  can  it  be 
determined  at  present  whether  it  is  more  correctly  classified 
among  the  bacteria  or  among  the  protozoa. 

*  "Deutsche  Med.  Wochenschrift,"  1894,  xux. 

f  "Ann.  de  1'Inst.  Pasteur,"  1896,  488. 

J  "Journal  of  Infectious  Diseases,"  1906,  in,  148. 


480  Vincent's  Angina 

Cultivation. — The  organisms  were  cultivated  by  Ttmni- 
cliff  upon  the  surface  of  ascitic  fluid  agar-agar  (i  13)  under 
strictly  anaerobic  conditions  at  37°  C.  After  two  or  three 
days  the  fusiform  bacillus  appeared  in  the  form  of  delicately 
whitish  colonies,  0.5  to  2  mm.  in  diameter,  resembling  colonies 
of  streptococci.  By  transplanting  these,  pure  cultures  of 
Bacillus  fusiformis  were  obtained.  In  the  transplantation 
tubes  the  organism  again  grew  in  the  form  of  similar  whitish 
colonies,  a  flocculent  deposit  accumulating  at  the  bottom  of 
the  water  of  condensation. 

Loffler's  Blood-serum  Mixture. — After  twenty-four  to  forty- 
eight  hours  similar  colonies  appear  and  a  similar  flocculent 
deposit  collects  in  the  condensation  water. 

Rabbit's  Blood  Agar-agar. — The  growth  is  similar,  but 
brownish  in  color. 

Glycerin  Agar-agar. — No  growth. 

Glucose  Agar-agar  Stab. — A  delicate  whitish  growth  with 
small  lateral  prolongations  develops  along  the  path  of  the 
wire  in  twenty-four  to  forty-eight  hours.  Some  gas  is 
formed. 

Litmus  Milk. — In  forty-eight  hours  there  is  a  moderate 
growth.  The  litmus  becomes  decolorized.  There  is  no 
coagulation.  When  oxygen  is  admitted  the  medium  re- 
gains its  lost  color. 

Potato. — No  growth. 

Bouillon  and  Dextrin-free  Bouillon. — No  growth. 

Glucose-bouillon. — No  growth  when  more  than  i  per  cent,  of 
glucose  is  present.  The  medium  is  clouded  with  some  sedi- 
ment. 

From  all  of  the  cultures  a  somewhat  offensive  odor  is 
given  off. 

Morphology. — The  Bacillus  fusiformis  presents  the  same 
appearances,  no  matter  what  medium  it  grows  upon.  It 
measures  3  to  10  ^  in  length,  0.3  to  0.8  /w  in  thickness.  The 
greatest  diameter  is  at  the  center,  from  which  the  organisms 
gradually  taper  to  blunt  or  pointed  extremities. 

The  organisms  stain  with  Loffler's  alkaline  methylene-blue, 
with  diluted  carbol-fuchsin,  by  Gram's  method,  and  by 
Giemsa's  method.  The  staining  is  intense,  but  is  rarely  uni- 
form, the  substance  usually  being  interrupted  by  vacuoles 
or  fractures,  reminding  one  of  those  seen  in  the  diphtheria  and 
tubercle  bacilli.  The  organism  forms  endospores  sometimes 
situated  at  the  center,  but  more  frequently  toward  one  end. 


Morphology 


481 


In  twenty-four  to  forty-eight  hours  filaments  are  seen. 
These  are  of  the  same  diameter  throughout,  and  usually  con- 
tain deeply  staining  bodies,  sometimes  round,  oftener  in  bands. 


Fig.  154. — Bacillus  fusiformis.  Pure  culture  grown  forty-eight  hours 
anaerobically  on  Loffler's  blood-serum.  (Ruth  Tunnicliff  in  "Journal 
of  Infectious  Diseases.") 


Fig.  155. — Bacillus  fusiformis.  Pure  culture  grown  forty-eight  hours 
anaerobically  in  the  fluid  of  condensation  of  Loffler's  blood-serum. 
(Ruth  Tunnicliff  in  "  Journal  of  Infectious  Diseases.") 

Most  of  the  filaments  are  made  up  of  strings  of  bacilli,  but 
some  stain  uniformly.     About  the  fourth  or  fifth  day  the 
31 


482 


Vincent's  Angina 


spirals  make  their  appearance,  sometimes  in  enormous 
numbers.  As  a  rule,  they  stain  uniformly,  some  show  the 
dark  bodies  seen  in  the  bacilli  and  filaments.  They  form  from 


Fig.  156. — Bacillus  fusiformis.     Pure  culture  grown  four  days  in  ascites 
broth.     (Ruth  Tunnicliff  in  "  Journal  of  Infectious  Diseases.") 


Fig.   157. — Bacillus  fusiformis.     Smear  from   gum  in   normal  mouth. 
(Ruth  Tunnicliff  in  "Journal  of  Infectious  Diseases.") 


one  to  twenty  turns,  which  are  not  uniform.  The  spirals 
are  flexible,  the  ends  pointed.  The  spirals  persist  in  the 
cultures,  at  times  for  fifty-five  days. 


Pathogenesis  483 

Neither  the  bacilli  nor  the  spirals  show  any  progressive 
movement,  though  with  the  dark-field  illuminator  they  show 
a  slight  vibratile  and  rotary  movement.  No  flagella  were 
observed. 

Pathogenesis. — Pure  cultures  of  the  organisms  were  in- 
oculated into  guinea-pigs  without  result.  As  in  Vincent's 
angina  the  throat  always  contains  staphylococci  and  strep- 
tococci, and  not  infrequently  diphtheria  bacilli,  it  is  thought 
by  many  that  Bacillus  fusiformis  does  not  initiate  the  morbid 
process,  but  is  a  secondary  invader,  by  which  simpler  inflam- 
mations are  intensified  and  made  necrotic. 

This  seems  to  be  particularly  true  of  diphtheria,  and  may 
account  for  the  occurrence  of  noma,  in  which  gangrenous 
condition  of  the  mouth  and  genitals  the  organisms  have  been 
found  in  great  numbers. 


CHAPTER   XIII. 

THRUSH. 

OIDIUM  ALBICANS  (ROBIN). 

THRUSH,  Soor  (German),  Muguet  (French),  or  parasite 
stomatitis  is  an  affection  of  marasmatic  infants  and  adults 
characterized  by  the  occurrence  of  peculiar  whitish  patches 
upon  an  inflamed  oral  mucous  membrane.  The  white  of  the 
patches  consists  of  material  that  is  not  easily  removed,  but 
which  when  detached  leaves  a  bleeding  surface  upon  which  it 
forms  again.  Upon  microscopic  examination  the  white  sub- 
stance proves  to  be  composed  of  masses  of  mycelia  with  en- 
larged epithelial  cells  and  leukocytes.  The  affection  is  far 
more  frequent  in  children  than  in  adults.  It  seems  not  to 
occur  among  healthy  children,  but  among  those  suffering  from 
marasmus,  and  particularly  among  those  whose  mouths  have 
already  become  sore  through  neglect.  It  is  usually  confined 
to  the  mouth,  but  may  spread  to  the  pharynx,  to  the  larynx, 
in  rare  cases  to  the  esophagus,  in  very  rare  cases  to  the 
stomach  and  intestines,  and  in  exceptional  cases,  both  in 
adults  and  children,  may  become  a  generalized  disease  through 
hematogenous  distribution,  and  be  attended  by  mycotic  in- 
flammatory lesions  in  the  kidneys,  the  liver,  and  the  brain. 

The  specific  micro-organism  seems  to  have  been  discovered 
in  1839  by  Langenbeck*  and  Berg,  f  Langenbeck  missed  the 
significance  of  the  organism  altogether,  for,  finding  it  in  a 
case  of  typhoid  fever,  he  conceived  it  to  be  the  cause  of  that 
disease.  Berg,  on  the  other  hand,  regarded  it  as  the  cause 
of  the  thrush.  Robin  {  furnished  the  first  correct  description 
of  the  organism  and  gave  it  its  name,  O'idium  albicans.  Many 
systematic  writers  have  exercised  themselves  concerning  the 
exact  place  in  the  botanical  system  in  which  the  organisms 
should  be  placed.  Thus,  Gruby  and  Heim  regarded  it  as  a 

v     *  See  Kehrer,  "Ueber  den  Soorpilz,"  etc.,  Heidelberg,  1883. 

fSee  Behrend,  "Deutsche  med.  Wochenschrift,"  1890. 

|  "Histoire  naturelle  des  vegetaux  parasites  qui  croissent  sur  1'homme 
et  sur  les  animaux  vivants,"  Paris,  1853. 

484 


Morphology  485 

sporotrichum ;  Robin,  as  an  oidium;  Quinquaud,  as  a  syringo- 
spora;  Hallein  called  it  Stemphylium  polymorpha;  Grawitz, 
as  Mycoderma  vini;  Plaut,  as  Monilia  Candida;  Guidi,  Ress, 
Brebeck-Fischer,  as  a  saccharomyces ;  Laurent,  as  Dematium 
albicans;  Linossier  and  Roux,  as  a  mucor,  and  Alav,  Olsen, 
and  Vuillemin,  as  Endomyces  albicans.  The  matter  is  still 
undecided  and  until  it  is  finally  agreed  upon  it  seems  best  to 
resort  to  the  original  name,  Oidium  albicans. 

Morphology. — The  organism  consists  of  elements  that 
bear  a  close  resemblance  to  yeast  cells  and  multiply  by  bud- 
ding, of  hyphae  and  mycelial  threads  into  which  these  grow, 
and  of  chlamydospores  and  conidia. 


Fig.  158. — Oidium.     (Kolle  and  Wassermann.) 

The  yeast-like  elements  measure  5  to  6  [i  in  length  and  4  {i 
in  breadth.  They  have  an  oval  form  and  cannot  be  distin- 
guished from  yeast  cells.  The  mycelia  are  formed  by  elon- 
gation of  these  elements,  some  of  which  appear  slightly  elon- 
gate, some  greatly  elongate  and  slender  and  more  or  less 
septate,  like  those  of  the  true  molds.  They  are  refractile, 
doubly  contoured,  and  contain  droplets,  vacuoles,  and  gran- 
ules. In  the  interior  of  the  hyphae  conidia-like  organs  often 
appear,  and  chlamydospores  appear.  The  latter  are  large, 
oval,  doubly  contoured,  highly  refracting,  and  have  been  seen 
by  Plaut  to  germinate. 

The  morphology  is,  however,  extremely  varied,  and  the 
greatest  differences  of  interpretation  have  been  expressed  re- 
garding the  different  elements. 


486 


Thrush 


Cultivation. — The  organism  grows  readily  in  artificial 
media,  both  with  and  without  free  access  of  oxygen.  An  acid 
reaction  is  most  appropriate. 

Colonies. — The  superficial  colonies  upon  gelatin  plates  are 
rounded,  waxy,  and  coarsely  granular.  The  deep  colonies  are 
irregular  in  shape  and  show  feathery  processes  extending  into 
the  medium.  The  color  varies  according  to  the  composition 
of  the  medium,  from  snow  white  on  ordinary  gelatin  to  meat- 
red  on  beet-root  gelatin.  A  sour  odor  is 
given  off  from  the  cultures. 

Gelatin  Punctures. — Along  the  line  of 
puncture  there  is  a  slow  formation  of 
rounded,  feathery,  colorless  colonies,  not 
unlike  those  shown  by  many  molds. 
The  gelatin  is  slowly  liquefied  only  when 
it  contains  sugar.  In  such  cultures 
chlamydospores  are  abundant. 

Agar-agar. — Cultures  are  similar  to 
those  in  gelatin. 

Bouillon. — The  organism  grows  only  at 
the  bottom  of  the  tube  in  the  form  of 
yellowish- white  flocculi. 

Potato. — Various  in  different  cases. 
Often  floury. 

Milk. — The  organism  grows  very  poorly 
in  milk,  which  is  not  coagulated  or  fer- 
mented. 

Fermentation. — The  organism  utilizes 
dextrin,  mannite,  alcohol,  lactose,  and 
glycerin  without  fermentation.  Saccha- 
rose is  destroyed  without  invertin  for- 
mation. Glucose,  levulose,  and  maltose 
are  fermented  very  slowly. 

Metabolic  Products. — In  addition  to  the  ferments  that 
act  upon  the  sugars,  etc.,  and  soften  the  gelatin,  the  organ- 
ism forms  alcohol,  aldehyd,  and  acetic  acid. 

Pathogenesis. — Animals  are  not  known  to  suffer  from 
spontaneous  infection.  Grawitz  was  able  to  induce  thrush  in 
puppies.  Stooss  inoculated  the  scarified  vaginas  of  rabbits 
with  mixed  cultures  of  pyogenic  cocci  and  o'idium  and  ob- 
tained thrush  plaques.  The  oidi'um  alone  was  unable  to 
secure  a  foothold.  Doderlein,  Grosset,  and  Stooss  all  suc- 
ceeded in  producing  abscesses,  sometimes  by  subcutaneous 


Fig-  159. — Oidium 
albicans.  Culture 
in  gelatin  (Hansen). 


Immunity  487 

injection  of  the  oidium,  but  usually  only  when  it  was  combined 
with  pus  cocci.  In  such  abscesses  the  cocci  are  killed  off  by 
phagocytes,  and  when  cultures  are  made  only  the  oi'dium 
grows.  Plaut  points  out  that  this  is  exactly  the  reverse  of 
what  happens  in  artificial  cultures  of  the  two  organisms 
where  the  cocci  outgrow  and  kill  off  the  oidium. 

Intravenous  injection  sometimes  causes  generalized  oidium 
infection,  with  colonies  of  the  micro-organism  in  the  kidneys, 
heart-muscle,  peritoneum,  liver,  spleen,  stomach,  and  in- 
testines. The  central  nervous  system  may  also  show  small 
foci  of  the  infection. 

Immunity. — Roger*  and  Noissette  f  were  able  to  immunize 
animals  against  oidium. 

*"Compt.-rende  de  la  Societe  de  Biologic,"  Paris,  1896. 
t  "These  de  Paris,"  1898. 


CHAPTER   XIV. 

WHOOPING-COUGH. 

THE;  BORDET-GENGOU  BACILLUS. 

THE  subacute,  contagious,  undoubtedly  infectious  disease 
of  childhood,  characterized  by  periodic  attacks  of  spasmodic 
cough  and  laryngeal  spasm,  terminating  in  a  prolonged 
crowing  inspiration  and  frequently  followed  by  vomiting 
and  prostration,  known  as  pertussis,  or  whooping-cough,  has 
long  been  subject  to  bacteriologic  investigation.  Deichler, 
Kurloff,  Szemetzchenko,  Cohn,  Neumann,  Ritter,  and 
Afanassiew  have  all  written  upon  bacteria  which  they  sup- 
posed to  be  the  causal  factors  of  the  disease,  but  which  time 
has  consigned  to  oblivion.  Koplik*  and  Czaplewski  and 
Henself  described  micro-organisms  that  for  some  years  at- 
tracted attention  and  caused  more  or  less  discussion  as  to 
which  might  be  the  real  excitant  of  the  disease  or  whether 
they  were  identical  organisms.  As  time  passed,  both  obser- 
vations lacked  sufficient  confirmation  to  carry  conviction 
of  their  importance,  and  they,  too,  fell  into  oblivion.  A  still 
different  organism  was  described  by  VincenziJ,  but  also 
failed  to  meet  sufficient  confirmatory  evidence  to  prevent 
it  from  meeting  the  fate  of  its  predecessors. 

Spengler,§  Kraus  and  Jochmann,||  and  Davis**  showed  the 
frequent  presence  of  minute  bacilli  in  the  sputum  and  also  in 
the  lesions  of  the  disease.  They  were,  almost  beyond  doubt, 
influenza  bacilli. 

In  1906  Bordet  and  Gengouft  described  a  new  organism 
whose  importance  was  supported  by  such  weighty  evidence 

*  "Centralbl.  f.  Bakt.,"  etc.,  Sept.  15,  1897,  xxn,  8  and  9,  p.  222. 
t  "Deutsch.  med.  Wochenschrift,"  1897,  No.  57,  p.  586;  "  Centralbl. 
f.  Bakt.,"  etc.,  Dec.  22,  1897,  xxn,  Nos.  22  and  23,  p.  641. 

t"Alti  della  Accademia  di  Medicina  in  Torino,"  LXI,  5-7;  "Cen- 
tralbl. f.  Bakt.,"  etc.,  Jan.  19,  1898,  xxm,  p.  273. 
§"Deutsch.  med.  Wochenschrift,"  1897,  830. 
||  "Zeitschrift  fur  Hygiene,"  etc.,  1901,  xxxvi,  193. 
**  "Jour.  Infectious  Diseases,"  1906,  in,  i. 
ft  "Ann.  de  1'Inst.  Pasteur,"  1906,  xx,  731. 

488 


Staining  489 

as  the  formation  of  an  endotoxin  sufficiently  active  to  ex- 
plain the  symptoms,  and  the  fixation  of  complement  by  the 
serum  of  the  infected  animal.  This  organism,  therefore, 
presents  itself  as  sufficiently  meritorious  to  maintain  the 
field  for  the  present. 

Morphology. — The  organisms,  as  found  in  the  sputum,  oc- 
cur as  very  minute  ovoid  rods  of  about  the  same  size  as  the 
influenza  bacillus.  They  measure  approximately  1.5  fi  in 
length  by  0.3  u  in  breadth.  They  do  not  remain  united 
as  chains  or  rods,  but  separate  as  individuals.  They  are 


Fig.  1 60. — The  Bordet-Gengou  bacillus  of  whooping-cough.  Twenty- 
four-hour-old  culture  upon  solid  media  containing  blood  (Bordet- 
Gengou). 

somewhat  pleomorphous,  yet  the  variations  are  not  con- 
siderable. Involution  forms  are  not  common.  There  are 
no  spores,  no  flagella,  no  motility. 

Staining. — The  organisms  do  not  hold  the  stain  well. 
Most  of  the  bacilli  are  pale,  some  contain  uncolored  areas 
or  vacuoles.  In  some  cases  the  ends  of  the  bacilli  appear 
more  deeply  stained  than  the  middle.  They  do  not  stain 
by  Gram's  method.  The  discoverers  recommend  that  the 
organism  be  stained  with — 

Toluidin  blue 5  ^  Dissolve  and  add  500  of  5  per  cent. 

Alcohol 100  X      aqueous  carbolic  acid.     After  two 

Water 500  j       days  filter. 

Isolation. — The  organisms  occur  in  almost  pure  cultures 
in  the  whitish  expectoration  which  escapes  from  the  bronchi 


49°  Whooping-Cough 

in  the  beginning  of  the  disease.     Later  they  become  few  and 
may  disappear,  though  the  symptoms  of  the  disease  persist. 
Cultivation. — The  cultures  were  secured  upon  a  special 
medium  made  as  follows: 


I. 


l}**.  Pour  off  the  fluid. 


II.    Potato  extract  (made  as  above) .  .    50  c.c.  ]  Boil,  dissolve,  filter, 

0.6  per  cent,  aqueous  NaCl 150  c.c.  >•      and  tube;  2  to  3 

Agar-agar 5  gm.  J      c.c.  to  a  tube. 

III.  To  each  tube  add  an  equal  volume  of  defibrinated  rabbits'  (or, 
better,  human)  blood  before  cooling  to  the  point  of  coagulation.  Permit 
the  tubes  to  solidify  in  the  oblique  position. 

At  first  the  growth  is  scant,  but  upon  transplantation 
grows  better  and  better,  until  finally  it  may  be  made  to 
grow  upon  other  media,  such  as  blood-agar,  ascitic  agar,  or 
broth  to  which  blood  or  ascitic  fluid  has  been  added.  The 
organism  is  a  strict  aerobe.  It  grows  best  at  37°  C.,  but  also 
grows  at  temperatures  as  low  as  5°  to  10°  C.  On  appropriate 
culture-media  Wollstein  found  it  might  remain  alive  for  two 
months. 

Metabolic  Products. — An  endotoxin  was  found  by  Bordet 
and  Gengou,  the  method  of  preparing  which  was  improved 
by  Besredka*  as  follows:  The  growth  upon  agar-agar  is 
removed  with  a  small  quantity  of  salt  solution,  dried  in 
vacuo,  and  ground  in  a  mortar  with  a  small  measured 
quantity  of  salt.  Enough  distilled  water  is  then  added  to 
make  a  0.75  per  cent,  solution,  after  which  the  mixture  is 
centrifugalized  and  decanted.  Of  this  preparation  i  to  2 
c.c.  usually  killed  a  rabbit  about  twenty-four  hours  after 
intravenous  injection.  Subcutaneous  injection  caused  a 
necrosis  without  suppuration  and  without  constitutional 
symptoms.  Small  quantities  of  the  toxin  placed  in  the 
rabbit's  eye  caused  local  necrosis,  with  little  inflammatory 
reaction.  The  introduction  of  dead  or  living  cultures  into 
the  peritoneal  cavity  of  guinea-pigs  caused  death  with  great 
effusion  and  hemorrhage  in  the  peritoneal  tissues. 

Pathogenesis. — Inoculation  of  monkeys  with  cultures  of 
the  bacillus  failed  to  produce  the  disease.  Klimenko,t 
however,  succeeded  in  infecting  monkeys  and  pups  by  intra- 
tracheal  introduction  of  pure  cultures.  After  a  period  of 

*  Bordet,  "Bull,  de  la  Soc.  Roy.  de  Bruxelles,"  1907. 
t  "Centralbl.  f.  Bakt.,"  etc.  (Orig.),  XLVIII.  64. 


Pathogenesis  491 

incubation  an  illness  came  on,  the  most  marked  symptoms 
being  pyrexia  and  pulmonary  irritation.  After  two  or  three 
weeks  the  dogs  died.  Postmortem  examination  showed 
catarrh  of  the  respiratory  tissues  with  patches  of  broncho- 
pneumonia.  Healthy  dogs  contracted  the  disease  by  con- 
tact with  those  suffering  from  the  infection.  Frankel*  ob- 
tained similar  results. 

The  differences  between  the  Bordet-Gengou  bacillus  and 
the  influenza  bacillus  are  not  great.  In  size,  mode  of  oc- 
currence, grouping  and  staining  there  is  much  resemblance, 
between  the  two.  Culturally,  however,  they  differ  because 
the  influenza  bacillus  grows  best  upon  hemoglobin  or  blood 
agar-agar,  which  is  less  adapted  for  the  isolation  of  the  Bor- 
det-Gengou bacillus  than  the  culture-medium  given  for  its 
cultivation,  upon  which  the  influenza  bacillus  does  not 
grow  well.  Further,  we  have  as  differential  the  peculiar 
endotoxin  of  the  Bordet-Gengou  bacillus,  the  successful 
infection  of  dogs  and  monkeys  with  the  disease  resembling 
whooping-cough,  and  the  transmission  of  this  infection 
from  animal  to  animal  by  natural  means. 

The  subject  of  complement  deviation  as  a  proof  of  the 
specific  nature  of  the  organism  is  still  under  consideration. 
Bordet  and  Gengou  found  that  the  serum  of  convalescent 
patients  fixed  complement  when  applied  to  the  bacilli; 
Frankel  and  Wollstein,f  that  it  did  not.  It  is  claimed  by 
Bordet  and  Gengou  that  the  difference  in  results  came  about 
through  the  employment  of  different  culture-media  in  per- 
forming the  Complement  fixation  tests. 

*  "Mtinchener  med.  Wochenschrift,"  1908,  p.  1683. 
t  "Journal  of  Exp.  Med.,"  1909,  xi,  41. 


CHAPTER   XV. 

PNEUMONIA. 
LOBAR  OR  CROUPOUS  PNEUMONIA, 

DIPLOCOCCUS  PNEUMONIA  (WEICHSEXBAUM). 

General  Characteristics.— A  minute,  spheric,  slightly  elongate 
or  lancet-shaped,  non-motile,  non-flagellate,  non-sporogenous,  aerobic 
and  optionally  anaerobic,  non-chromogenic,  non-liquefying  diplococ- 
cus,  pathogenic  for  man  and  the  lower  animals,  staining  by  ordinary 
methods  and  by  Gram's  method. 

"  Pneumonia,"  while  generally  understood  to  refer  to  the 
lobar  form  of  the  disease  particularly  designated  as  croupous 
pneumonia,  is  a  vague  term,  comprehending  a  number  of  quite 
dissimilar  inflammatory  conditions  of  the  lung.  This  being 
true,  no  single  micro-organism  can  be  "  specific  "  for  all. 
Indeed,  pneumonia  must  be  conceived  of  as  a  group  of  dis- 
eases, and  the  various  micro-organisms  associated  with  it 
must  be  separately  considered  in  connection  with  the 
particular  varieties  of  the  disease  in  which  they  occur. 

The  micro-organism,  that  can  be  demonstrated  in  at  least 
75  per  cent,  of  cases  of  lobar  pneumonia,  which  is  almost 
universally  accepted  to  be  the  cause  of  the  disease,  and  about 
whose  specificity  very  few  doubts  can  now  be  raised,  is  the 
Diplococcus  pneumoniae,  or  pneumococcus  of  Frankel  and 
Weichselbaum. 

Priority  of  discovery  of  the  pneumococcus  seems  to  be  in 
favor  of  Sternberg,*  who  as  early  as  1880  described  an 
apparently  identical  organism  which  he  secured  from  his 
own  saliva.  Pasteur  |  seems  to  have  cultivated  the  same 
micro-organism,  also  from  saliva,  in  the  same  year.  The 
researches  of  the  observers  whose  names  are  now  attached 
to  the  organism  were  not  completed  until  five  years  later. 
It  is  to  Telamon,  f  Frankel,§  and  particularly  to  Weichsel- 

*  ''National  Board  of  Health  Bulletin,"  1881,  vol.  n. 
t  "Compte-rendus  Acad.  des  Sciences,"  1881,  xcn,  p.  159. 
t  "Societe  anatom.  de  Paris,"  Nov.  30,  1883. 
§  "Deutsche  med.  Wochenschrift,"  1885,  31. 
492 


Morphology  493 

baum,*  however,  that  we  are  indebted  for  the  discovery  of 
the  relation  which  the  organism  bears  to  pneumonia. 

Distribution. — The  pneumococcus  is  one  of  a  group  of 
widely  disseminated  organisms  of  the  respiratory  tract.  It 
is  characterized  by  certain  peculiarities  of  morphology, 
certain  metabolic  peculiarities,  a  definite  pathogenesis,  and 
a  distinct  agglutinative  reaction  with  immune  serum. 
From  such  typical  individuals  a  number  of  irregular  depart- 
ures are  known,  and  recent  researches  make  it  certain  that 
many  of  the  organisms  formerly  looked  upon  as  pneumococci 
are  different  and  perhaps  harmless.  The  pneumococcus 
is  a  purely  parasitic,  pathogenic  organism,  best  known  to 
us  in  its  relation  to  croupous  pneumonia,  where  it  is  present 
in  the  lungs,  sputum,  and  blood.  It  may  be  found  in  the 
saliva  of  a  large  number  of  healthy  persons,  Parke  and 
Williams,  f  especially  during  the  winter  months,  Longcope 
and  Fox,  J  and  the  inoculation  of  human  saliva  into  rabbits 
frequently  causes  septicemia  in  which  the  pneumococci  are 
abundant  in  the  blood  and  tissues.  Its  frequent  occurrence 
in  the  saliva  led  Flugge  to  describe  it  as  Bacillus  septicus 
sputigenus.  It  is  occasionally  found  in  inflammatory  lesions 
other  than  pneumonia,  as  will  be  pointed  out  below. 

Morphology. — The  organism  is  variable  in  morphology. 
When  grown  in  bouillon  it  appears  oval,  has  a  pronounced 
disposition  to  occur  in  pairs,  and  not  infrequently  forms 
chains  of  five  or  six  members,  so  that  some  have  been  dis- 
posed to  look  upon  it  as  a  streptococcus  (Gamaleia) .  In  the 
fibrinous  exudate  from  croupous  pneumonia,  in  the  rusty 
sputum,  and  in  the  blood  of  rabbits  and  mice  containing 
them,  the  organisms  occur  in  pairs,  have  a  lanceolate  shape, 
the  pointed  ends  usually  being  approximated,  and  are 
usually  surrounded  by  a  distinct  halo  or  capsule  of  clear, 
colorless,  homogeneous  material,  thought  by  some  to  be  a 
swollen  cell- wall,  by  others  a  mucus-like  secretion  given  off 
by  the  cells.  When  grown  in  culture-media,  especially 
upon  solid  media,  the  capsules  are  not  apparent.  The 
elongate  form  has  led  Migula§  to  describe  it  under  the  name 
Bacterium  pneumoniae. 

The  organism  measures  about  i  ^  in  greatest  diameter, 

*  "Wiener  med.  Jahrbuch,"  1886,  p.  483. 

t  "Jour.  Exp.  Med.,"  vn,  Aug.  7,  1905,  p.  403. 

t  Ibid.,  p.  430. 

§  "System  der  Bakterien,"  Jena,  1900,  p.  347. 


494  Pneumonia 

is  without  motility,  has  no  flagella,  forms  no  spores,  and 
seems  unable  long  to  resist  unfavorable  conditions  when 
grown  artificially. 

Staining. — It  stains  well  with  the  ordinary  solutions  of 
the  anilin  dyes,  and  gives  most  beautiful  pictures  in  blood 
and  tissues  when  stained  by  Gram's  and  Weigert's  methods. 

To  demonstrate  the  capsules,  the  glacial  acetic  acid  method 
of  Welch*  may  be  used.  The  cover-glass  is  spread  with 
a  thin  film  of  the  material  to  be  examined,  which  is  dried 
and  fixed  as  usual.  Glacial  acetic  acid  is  dropped  upon  it 


Fig.  161. — Capsulated  pneumococci  in  blood  from  the  heart  of  a  rabbit; 
carbol-fuchsin,  partly  decolorized.      X  1000. 


for  an  instant,  poured  (not  washed)  off,  and  at  once  followed 
by  anilin-water  gentian  violet,  in  which  the  staining  con- 
tinues several  minutes,  the  stain  being  poured  off  and 
replaced  several  times  until  the  acid  has  all  been  removed. 

Finally,  the  preparation  is  washed  in  water  containing  i  or 
2  per  cent,  of  sodium  chlorid,  and  may  be  examined  at  once 
in  the  salt  solution,  or  mounted  in  balsam  after  drying. 
The  capsules  are  more  distinct  when  the  examination  is 
made  in  water. 

Hissf  recommends  the  following  as  an  excellent  method 

*  "Bull,  of  the  Johns  Hopkins  Hospital,"  Dec.,  1892,  p.  128. 

t  Abstract,  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Bd.  xxxi,  No.  10,  p. 
302,  March  24,  1902.  More  complete  details  appear  in  a  later  paper  in 
the  "Journal  of  Experimental  Medicine,"  vi,  p.  338. 


Isolation  495 

of  staining  the  capsules  of  the  pneumococcus :  The  organism 
is  first  cultivated  upon  ascites  serum-agar  to  which  i  per 
cent,  of  glucose  is  added.  The  drop  containing  the  bacteria 
to  be  stained  is  spread  upon  a  cover-glass  mixed  with  a 
drop  of  serum  or  a  drop  of  the  fluid  culture-medium,  and 
dried  and  fixed.  A  half-saturated  aqueous  solution  of  gen- 
tian violet  is  applied  for  a  few  seconds  and  then  washed 
off  in  a  25  per  cent,  solution  of  carbonate  of  magnesium. 
The  preparation  is  then  mounted  in  a  drop  of  the  latter 
solution  and  examined. 

If  it  is  desired  to  stain  the  capsules  and  preserve  the 
specimens  permanently  in  balsam,  Hiss  employs  a  5  or  10 
per  cent,  solution  of  fuchsin  or  gentian  violet  (5  c.c.  saturated 
alcoholic  solution  of  dye  in  95  c.c.  of  distilled  water).  The 
stain  is  applied  to  the  fixed  specimen  and  heated  until  it 
begins  to  steam,  when  the  stain  is  washed  off  in  a  20  per 
cent,  solution  of  crystals  of  sulphate  of  copper.  The  prepara- 
tion is  then  dried  and  mounted  in  balsam. 

Hiss  finds  this  stain  a  useful  aid  in  differentiating  the 
pneumococcus  from  the  streptococcus,  with  which  it  is 
easily  confounded  if  the  capsules  are  not  distinct,  and  to 
which  it  is  probably  closely  related. 

To  demonstrate  the  organisms  in  sections  of  tissue  either 
Gram's  or  Weigert's  methods  may  be  used,  with  beautiful 
results. 

Isolation. — When  desired  for  purposes  of  study,  the 
pneumococcus  may  be  obtained  by  inoculating  rabbits  with 
pneumonic  sputum  and  recovering  the  organisms  from  the 
heart's  J^lood,  or  it  may  be  obtained  from  the  rusty  sputum 
of  pneumonia  by  the  method  employed  by  Kitasato  for 
securing  tubercle  bacilli  from  sputum:  A  mouthful  of  fresh 
sputum  is  washed  in  several  changes  of  sterile  water  to  free 
it  from  the  bacteria  of  the  mouth  and  pharynx,  carefully 
separated,  and  a  minute  portion  from  the  center  transferred 
to  an  appropriate  culture- medium. 

Buerger,*  in  conducting  a  research  upon  pneumococcus 
and  allied  organisms  with  reference  to  their  occurrence  in  the 
human  mouth,  under  the  auspices  of  the  Rockefeller  Insti- 
tute, used  a  2  per  cent,  glucose-agar  of  a  neutral,  or,  at  most, 
0.5  per  cent,  phenolphthalein  acid  titer. 

*  "Jour.  Exp.  Med.,"  Aug.  25,  1905,  vn,  No.  5. 


496  Pneumonia 

"The  medium  was  usually  made  from  meat  infusion  and  contained 
1.5  to  2  per  cent,  peptone  and  2.4  per  cent.  agar.  Stock  plates  of 
these  media  (serum-agar  and  2  per  cent,  glucose-serum-agar)  were 
poured.  The  agar  or  glucose-agar  was  melted  in  large  tubes  and 
allowed  to  cool  down  to  a  temperature  below  the  coagulation  point  of 
the  serum.  One-third  volume  of  rich  albuminous  ascitic  fluid  was 
added,  and  the  resulting  media  poured  into  Petri  plates.  These  were 
tested  by  incubation  and  stored  in  the  ice-chest  ready  for  use.  .  .  . 

"The  plan  finally  adopted  [for  inoculating  the  plates]  was  as  fol- 
lows: A  swab  taken  from  the  mouth  was  thoroughly  shaken  in  a  tube 
of  neutral  bouillon.  From  this  primary  tube  dilutions  in  bouillon 
with  four,  six,  and  eight  loops  may  be  made.  A  small  portion  of  the 
dilute  mixture  was  poured  at  a  point  near  the  periphery  of  the  pre- 
pared plates.  By  a  slight  tilting  motion  the  fluid  was  carefully  dis- 
tributed over  -the  whole  surface  of  the  plates.  Care  must  be  taken 
to  avoid  an  excess  of  fluid.  It  was  found  that  plates  made  in  this 
way  gave  a  sufficiently  thick  and  discrete  distribution  of  surface  col- 


Cultivation. — The  organism  grows  upon  all  the  culture- 
media  except  potato,  but  only  between  the  temperature 
extremes  of  24°  and  42°  C.,  the  best  development  being  at 
about  37°  C.  The  growth  is  always  meager,  probably  be- 
cause of  the  metabolic  formation  of  formic  acid.  The  addi- 
tion of  alkali  to  the  culture-medium  favors  the  growth  of 
the  pneumococcus  by  neutralizing  this  acid.  Hiss  and 
Zinnser*  advise  that  the  culture-media  used  for  the  pneumo- 
coccus be  made  with  3  to  4  per  cent,  of  peptone. 

Colonies. — The  colonies  which  develop  at  24°  C.  upon 
gelatin  plates  (15  per  cent,  of  gelatin  should  be  used  to 
prevent  melting  at  the  temperature  required)  are  described 
as  small,  round,  circumscribed,  finely  granular  white  points 
which  grow  slowly,  never  attain  any  considerable  size, 
and  do  not  liquefy  the  gelatin. 

If  agar-agar  be  used  instead  of  gelatin,  and  the  plates 
kept  at  the  temperature  of  the  body,  the  colonies  appear 
transparent,  delicate,  and  dewdrop-like,  scarcely  visible  to 
the  naked  eye,  but  under  the  microscope  appear  distinctly 
granular,  a  dark  center  being  surrounded  by  a  paler  marginal 
zone. 

Upon  the  medium  recommended  by  Buerger  for  isolating 
the  pneumococcus,  the  colonies  appear  in  from  eighteen  to 
twenty-four  hours,  the  surface  colonies  being  circular  and 
disk-like.  When  viewed  from  above,  the  surface  appears 
glassy  with  a  depressed  center.  When  viewed  from  the  side 
or  by  transmitted  light,  they  appear  as  distinct  milky  rings 
with  a  transparent  center.  This  "  ring  type  "  is  regarded  as 

*  "Text-book  of  Bacteriology,"  1910,  p.  356. 


Vital  Resistance  497 

characteristic  and  enables  the  organism  to  be  separated 
without  difficulty  from  the  streptococcus. 

Gelatin  Punctures. — In  gelatin  puncture  cultures,  made 
with  15  instead  of  the  usual  10  per  cent,  of  gelatin,  the 
growth  takes  place  along  the  entire  puncture  in  the  form 
of  minute  whitish  granules  distinctly  separated  from  one 
another.  The  growth  in  gelatin  is  always  very  meager. 

Agar-agar  and  Blood-serum. — Upon  agar-agar  and  blood- 
serum  the  growth  consists  of  minute,  transparent,  semi- 
confluent,  colorless,  dewdrop-like  colonies,  which  die  before 
attaining  a  size  which  permits  of  their  being  seen  without 
careful  inspection.  Upon  glycerin  agar-agar  the  growth  is 
more  luxuriant.  The  addition  of  a  very  small  percentage 
of  blood-serum  greatly  facilitates  the  growth. 

Bouillon. — In  bouillon  the  organisms  grow  well,  slightly 
clouding  the  medium.  With  the  death  of  the  organisms  and 
their  sedimentation,  the  medium  clears  again  after  a  few 
days. 

Milk. — Milk  is  an  appropriate  culture-medium,  its  casein 
being  coagulated.  Alkaline  litmus  milk  is  slowly  acidified. 

Potato. — The  pneumococcus  does  not  grow  upon  potato.* 

Vital  Resistance. — The  organism  usually  dies  after  a  few 
days  of  artificial  cultivation,  and  so  must  be  transplanted 
every  three  or  four  days.  In  rabbit's  blood,  in  sealed  tubes 
kept  cold,  it  can  sometimes  be  kept  alive  for  several  weeks. 
Hiss  and  Zinnserf  find  that  when  the  organism  is  planted  in 
"  calcium-carbonate-infusion  broth"  and  kept  in  the  ice-chest, 
the  cultures  often  remain  alive  for  several  months.  Bordoni- 
UffreduzziJ  found  that  when  pneumococci  were  dried  in 
sputum  attached  to  clothing,  and  were  exposed  freely  to  the 
light  and  air,  they  retained  their  virulence  for  rabbits  for 
from  nineteen  to  ninety-five  days.  Direct  sunlight  destroyed 
their  virulence  in  twelve  hours.  Guarniere§  found  that  dried 
blood  containing  pneumococci  remained  virulent  for  months. 

The  pneumococcus  is  destroyed  in  ten  minutes  by  a  tem- 
perature of  52°  C.  It  is  highly  sensitive  to  all  disinfectants, 
weak  solutions  quickly  killing  it. 

*  Ortmann  asserts  that  the  pneumococcus  can  be  grown  on  potato  at 
37°  C.,  but  this  is  not  generally  confirmed.  The  usual  acid  reaction  of 
the  potato  would  indicate  that  it  was  a  very  unsuitable  culture-medium. 

t  Loc.  cit. 

I  "Arch.  p.  1.  Sc.  Med.,"  1891,  xv. 

§  "Atti  della  R.  Acad.  Med.  di  Roma,"  1888,  iv. 
32 


498  Pneumonia 

Metabolic  Products. — Hiss  *  found  that  the  pneumococcus 
produces  acid  with  ease  from  monosaccharids,  disaccharids, 
and  such  complex  saccharids  as  dextrin,  glycogen,  starch, 
and  inulin.  The  fermentation  of  inulin  is  most  important  as 
a  means  of  differentiating  pneumococci  from  streptococci, 
which  cannot  ferment  it. 

Toxic  Products. — Nothing  definite  is  known  about  the 
metabolic  toxic  products  of  the  pneumococcus.  That  the 
symptoms  of  pneumonia  are  not  entirely  dependent  upon 
the  disturbance  of  respiration  is  clearly  shown  by  the  fact 
that  the  patients  suffer  from  high  fever  and  have  marked 
leukocytosis  with  enlargement  of  the  spleen.  The  cases  in 
which  the  cocci  invade  the  blood  are  usually  more  serious 
than  those  in  which  their  operations  are  restricted  to  the 
lung. 

The  toxin  must  be  purely  or  almost  purely  intracellular, 
however,  as  filtered  cultures  are  scarcely  at  all  toxic. 

Auldf  found  that  if  a  thin  layer  of  prepared  chalk  were 
placed  upon  the  bottom  of  the  culture-glass,  it  neutralized 
the  lactic  acid  produced  by  the  pneumococcus,  and  enabled 
it  to  grow  better  and  produce  much  stronger  toxin.  Mal- 
fadyenj  found  that  by  freezing  cultures  of  the  pneumococcus 
with  liquid  air  and  then  destroying  them  by  trituration  in  the 
frozen  state  and  then  extracting  the  fragments  with  i :  1000 
caustic  potash  solution,  a  toxin  whose  activity  corresponded 
fairly  well  with  the  virulence  of  the  culture  could  be  secured. 
This  toxin  killed  rabbits  and  guinea-pigs  in  doses  varying 
from  0.5  to  i  c.c. 

Pathogenesis. — If  a  small  quantity  of  a  pure  culture 
of  the  virulent  organism  be  introduced  into  a  mouse,  rabbit, 
or  guinea-pig,  the  animal  dies  in  one  or  two  days.  Exactly 
the  same  result  can  be  obtained  by  the  introduction  of  a  piece 
of  the  lung-tissue  from  croupous  pneumonia,  by  the  intro- 
duction of  some  of  the  rusty  sputum,  and  frequently  by  the 
introduction  of  human  saliva.  Postmortem  examination 
of  infected  animals  shows  an  inflammatory  change  at  the 
point  of  subcutaneous  inoculation,  with  a  fibrinous  exudate 
similar  to  that  succeeding  subcutaneous  inoculation  with 
the  diphtheria  bacillus.  At  times,  and  especially  in  dogs, 
a  little  pus  may  be  found.  The  spleen  is  enlarged,  firm,  and 

*  "Jour.  Exp.  Med.,"  vn,  No.  5,  Aug.  25,  1905. 
f  "Brit.  Med.  Jour.,"  Jan.  20,  1900. 
J  Ibid.,  1906,  ii. 


Pathogenesis  499 

red-brown.  The  blood  with  which  the  cavities  of  the  heart 
are  filled  is  firmly  coagulated,  and,  like  that  in  other  organs 
of  the  body,  contains  large  numbers  of  the  bacteria,  most  of 
which  exhibit  a  lanceolate  form  and  have  distinct  capsules. 
The  disease  is  thus  shown  to  be  a  bacteremia  unassociated 
with  conspicuous  tissue  changes. 

In  such  cases  the  lungs  show  no  consolidation.  Even  if 
the  inoculation  be  made  by  a  hypodermic  needle  plunged 
through  the  breast-wall  into  the  pulmonary  tissue,  pneu- 
monia rarely  results.  Gamaleia*  reported  that  pneumonic 
consolidation  of  the  lungs  of  dogs  and  sheep  could  be  brought 
about  by  injecting  the  pneumococcus  through  the  chest- 
wall  into  the  lung.  Tchistowitsch  f  stated  that  by  intra- 
tracheal  injections  of  cultures  into  dogs  he  succeeded  in 
producing  in  7  out  of  19  experiments  typical  pneumonic 
lesions.  Monti  {  claimed  to  have  found  that  a  characteristic 
croupous  pneumonia  results  from  the  injection  of  cultures 
into  the  trachea  of  susceptible  animals.  A  very  interesting 
review  of  the  literature  of  the  experimental  aspects  of  the 
subject,  embracing  198  references,  will  be  found  in  Wads- 
worth's  paper  upon  "  Experimental  Studies  on  the  Etiology 
of  Acute  Pneumonitis."§ 

The  final  proof  that  true  pulmonary  consolidation,  i.  e., 
pneumonia,  can  be  produced  experimentally  by  cultures  of 
the  pneumococcus  is  to  be  found  in  a  paper  by  Lamar  and 
Meltzer.||  These  investigators  etherized  dogs,  kept  the 
mouth  open  by  means  of  a  large  wooden  gag,  drew  the  tongue 
forward  by  means  of  hemostatic  forceps,  and  then,  seizing  the 
median  glosso-epiglottic  fold,  pulled  it  forward  so  that  the 
posterior  aspect  of  the  epiglottis  presented  an  inclined 
plane.  Into  this  concavity  one  end  of  a  tube  is  placed. 
Under  the  protection  of  the  left  index-finger  the  tube  was 
directed  into  the  larynx  and  pushed  down  slowly  and  gently 
through  the  trachea  until  a  resistance  was  met  with.  The 
inner  end  of  the  tube  was  then  found  to  engage  in  a  bronchus 
— usually  the  right  bronchus.  A  pipet  containing  a  liquid 
culture  of  the  pneumococcus  was  next  attached  to  the 
external  end  of  the  tube,  and  by  means  of  a  syringe  the 

*  "Ann.  de  1'Inst.  Pasteur,"  1888,  u,  440. 

t  Ibid.,  1890,  m,  285. 

J  "Zeitschrift  fur  Hygiene,"  etc.,  1892,  xi,  387. 

\  "Amer.  Med.  Jour.  Sciences,"  1904,  cxxvn,  p.  851. 

||  "Jour.  Exp.  Med.,"  1912,  xv,  No.  2,  p.  133. 


500 


Pneumonia 


culture  (about  6  c.c.)  was  injected  into  the  bronchus.  The 
syringe  was  then  removed,  the  piston  withdrawn,  and  the 
syringe  again  attached  to  the  pipet.  By  the  injection  of 
air  the  culture  was  driven  deeper  into  the  bronchi.  The  tube 
was  then  clamped  and  withdrawn  and  the  animal  released. 
By  these  means  experimental  pneumonia,  with  the  typical 


Fig.  162. — Lung  of  a  child,  showing  the  appearance  of  the  organ  in 
the  stage  of  red  hepatization  of  croupous  pneumonia.  The  pneu- 
monia has  been  preceded  by  chronic  pleuritis,  which  accounts  for  the 
thickened  fibrous  trabeculae  extending  into  the  tissue,  and  which  may 
have  had  something  to  do  with  the  peculiarly  prominent  appearance 
of  the  bronchioles  throughout  the  lung. 

consolidation  and  lobar  distribution,  was  produced  in  42 
successive  cases.  The  course  of  the  inflammatory  disturb- 
ance thus  produced  was  rapid,  and  in  one  case  nearly  com- 
plete consolidation  had  occurred  in  seven  hours. 


Pathogenesis  501 

Lesions. — The  lesions  of  croupous  pneumonia  of  man  are 
almost  too  well  known  to  need  descripion.  The  distribution 
of  the  disease  conforms  more  or  less  perfectly  to  the  divisions 
of  the  lung  into  lobes,  one  or  more  lobes  being  affected. 
An  entire  lung  may  be  affected,  though,  as  a  rule,  the  apex 
escapes  consolidation  and  is  simply  congested.  The  in- 
vaded portion  of  the  lung  is  supposed  to  pass  through  a 
succession  of  stages  clinically  described  as  (i)  congestion, 
(2)  red  hepatization,  (3)  gray  hepatization,  and  (4)  resolu- 
tion. In  the  first  stage  bloody  serum  is  poured  out  into  the 
air-cells,  filling  them  with  a  viscid  reddish  exudate.  In 
the  second  stage  this  coagulates  so  that  the  tissue  becomes 
solid,  airless,  and  approximately  like  liver  tissue  in  appear- 
ance. The  third  stage  is  characterized  by  dissolution  of 
the  erythrocytes  and  invasion  of  the  diseased  air-cells  by 
leukocytes,  so  that  the  color  of  the  tissue  changes  from 
red  to  gray.  At  the  same  time  the  coagulated  exudate  be- 
gins to  soften  and  leave  the  air-cells  by  the  natural  pas- 
sages, and  the  stage  of  resolution  begins. 

In  more  rare  cases  circumscribed  areas  of  consolidation 
occur  in  the  lung  tissue.  The  inflammatory  lesions  of  other 
organs  present  nothing  characteristic  by  which  they  can  be 
recognized  by  macroscopic  examination. 

The  pneumococcus  is  not  infrequently  discovered  in  dis- 
eased conditions  other  than  croupous  pneumonia;  thus,  Foa, 
Bordoni-Uffreduzzi,  and  others  found  it  in  cerebrospinal 
meningitis;  Frankel,  in  pleuritis;  Weichselbaum,  in  perito- 
nitis; Banti,  in  pericarditis;  numerous  observers,  in  acute 
abscesses;  Gabbi  isolated  it  from  a  case  of  suppurative 
tonsillitis;  Axenfeld  observed  an  epidemic  of  conjunctivitis 
caused  by  it;  Zaufal,  Levy,  and  Schroder  and  Netter  have 
been  able  to  demonstrate  it  in  the  pus  of  otitis  media,  and 
Fouler  ton  and  Bonney*  isolated  it  from  a  case  of  primary 
infection  of  the  puerperal  uterus.  It  has  also  been  found 
in  arthritis  following  pneumonia,  and  in  primary  arthritis 
without  previous  pneumonia  by  Howard.f 

Interesting  statistics  concerning  the  relative  frequency  of 
pneumococcus  infections  in  adults  given  by  Netter  {  are  as 
follows : 

*  "Trans.  Obstet.  Soc.  of  London,"  1903,  part  n,  p.  128. 
t  "Johns  Hopkins  Hospital  Bulletin,"  Nov.,  1903. 
$  "Compte-rendu,"  1889. 


502  Pneumonia 

Pneumonia 65.95 

Bronchopneumonia 15.85 

Meningitis 13.00 

Empyema 8.53 

Otitis  media 2.44 

Endocarditis 1.22 

Hepatic  abscess 1.22 

In  46  consecutive  pneumococcus  infections  of  children 
he  found : 

Otitis  media 29 

Bronchopneumonia 12 

Meningitis 2 

Pneumonia i 

Pleurisy : i 

Pericarditis i 

Susceptibility. — Not  all  animals  are  equally  susceptible  to 
the  action  of  the  pneumococcus.  Mice  and  rabbits  are 
highly  sensitive,  dogs,  guinea-pigs,  cats,  and  rats  are  much 
less  susceptible,  though  they  may  also  succumb  to  the  in- 
oculation of  large  doses. 

Specificity. — The  etiologic  relationship  of  the  pneumo- 
coccus to  pneumonia  is  based  chiefly  upon  the  frequency  of 
its  presence  in  croupous  pneumonia.  Netter*  found  it  82 
times  in  82  autopsies  upon  such  cases;  Klemperer,  21  times 
out  of  2 1  cases  studied  by  puncturing  the  lung  with  a  hypo- 
dermic syringe.  Weichselbaum  obtained  it  in  94  out  of  129 
cases;  Wolf,  in  66  out  of  70;  and  Pierce,  in  no  out  of  121 
cases.  In  about  5  per  cent,  of  the  cases  it  remains  localized 
in  the  respiratory  apparatus;  in  95  per  cent.,  it  invades  the 
blood.  An  interesting  paper  upon  this  subject  has  been 
written  by  E.  C.  Rosenow.f 

The  conditions  under  which  it  enters  the  lung  to  produce 
pneumonia  are  not  known.  It  is  probable  that  some  sys- 
temic depravity  is  necessary  to  establish  susceptibility,  and 
in  support  of  this  view  we  may  point  out  that  pneumonia  is 
very  frequent,  and  exceptionally  severe  and  fatal,  among 
drunkards,  and  that  it  is  the  most  frequent  cause  of  death 
among  the  aged.  Whether,  however,  any  particular  form  of 
vital  depression  is  necessary  to  predispose  to  the  disease, 
further  study  will  be  required  to  tell. 

Virulence. — Pneumococci  vary  greatly  in  virulence,  and 
rapidly  lose  this  quality  in  artificial  culture.  When  it  is 

*  "Compte-rendu,"  1889. 

f  "Jour.  Infectious  Diseases,"  1904,  i,  p.  280. 


Bacteriologic  Diagnosis  503 

desired  to  maintain  or  increase  the  virulence,  a  culture  must 
be  frequently  passed  through  animals.  Washbourn  found, 
however,  that  a  pneumococcus  isolated  from  pneumonic 
sputum  and  passed  through  one  mouse  and  nine  rabbits 
developed  a  permanent  virulence  when  kept  on  agar-agar 
so  made  that  it  was  not  heated  beyond  100°  C.,  and  alka- 
linized  4  c.c.  of  normal  caustic  soda  solution  to  each  liter 
beyond  the  neutral  point  determined  with  rosolic  acid. 
The  agar-agar  is  first  streaked  with  sterile  rabbit's  blood, 
then  inoculated.  The  cultures  are  kept  at  37.5°  C.  Ordi- 
narily pneumococci  seem  unable  to  accommodate  them- 


Fig.    163. — Diplococcus   pneumoniae.     Colony   twenty-four   hours   old 
upon  gelatin.      X  100  (Frankel  and  Pfeiffer). 

selves  to  a  purely  saprophytic  life,  and  unless  continually 
transplanted  to  new  media  die  in  a  week  or  two,  some- 
times sooner.  Lambert  found,  however,  that  in  Marmorek's 
mixture  (bouillon  2  parts  and  ascitic  or  pleuritic  fluid  i 
part)  the  organisms  would  sometimes  remain  alive  as  long 
as  eight  months,  preserving  their  virulence  during  the  entire 
time. 

Virulence  can  also  be  retained  for  a  considerable  time  by 
keeping  the  organisms  in  the  blood  from  an  infected  rabbit, 
hermetically  sealed  in  a  glass  tube,  on  ice. 

Bacteriologic  Diagnosis. — It  is  usually  unnecessary  to 
call  upon  the  bacteriologist  to  assist  in  making  the  diagnosis 


504  Pneumonia 

of  pneumonia.  If  necessary,  the  expectoration  can  be 
examined  by  the  methods  already  given  for  staining  the 
pneumococcus,  or  rabbits  may  be  inoculated  and  the  organ- 
ism recovered  from  the  blood.  Caution  must  be  exercised 
in  using  this  means  of  diagnosis,  however,  as  the  organ- 
ism sometimes  occurs  in  normal  saliva,  and  is  a  common 
associated  organism  in  tuberculosis  and  other  respiratory 
diseases.  Wadsworth*  has  been  able  to  show  that  agglu- 
tination reactions  can  be  obtained  by  concentrating  the 
pneumococci  in  isotonic  solution  and  adding  the  serum. 
The  method  does  not  seem  easily  applicable  for  diagnosis. 
Neufeld  f  and  Wadsworth  J  have  also  found  that  when 
rabbit's  bile  is  added  to  a  pneumococcus  culture  so  as  to  pro- 
duce lysis  of  the  organisms,  the  addition  of  pneumococcus- 
immune  serum  to  the  clear  fluid  so  obtained  results  in  a 
specific  precipitation.  This  seems  to  have  little  practical 
importance,  however,  for  purposes  of  diagnosis.  It  is, 
however,  of  some  importance  in  assisting  in  the  recognition 
of  the  pneumococcus  and  differentiating  it  from  the  strepto- 
coccus, for  when  the  latter  organisms  are  similarly  treated  no 
precipitate  takes  place. 

Buerger§  found  that  all  pneumococci,  irrespective  of 
source,  were  agglutinated  by  pneumococcus  immune  serum, 
that  such  serum  was  capable  of  agglutinating  various  pyo- 
genic  streptococci,  certain  atypical  organisms,  and  certain 
strains  of  Streptococcus  mucosus  capsulatus.  The  sera  of 
pneumonia  patients  varies  in  its  power  to  agglutinate  dif- 
ferent pneumococci;  some  strains  were  agglutinated,  others 
not.  The  sera  of  normal  individuals  and  of  normal  rab- 
bits possess  no  agglutinating  power  for  pneumococci,  the 
atypical  organisms,  certain  streptococci,  and  the  Strepto- 
coccus mucosus  capsulatus. 

As  pneumococci  sometimes  grow  in  chains  instead  of  in 
pairs,  and  as  the  capsules  are  not  always  more  distinct  than 
the  capsules  that  sometimes  surround  streptococci,  it  may 
be  necessary  to  resort  to  special  methods  of  cultivation  for 
the  final  determination  of  the  organism.  One  of  the  first 
to  be  recommended  is  the  use  of  the  blood-agar  plate,  to 

*  "Jour.  Med.  Research,"  vol.  x,  p.  228,  1904. 

t  "Zeitschrift  fiir  Hygiene,"  1902,  xi. 

J  Loc.  cit. 

§  "Jour.  Exp.  Med.,"  Aug.  25,  1905,  vn,  No.  5. 


Immune  Serum  505 

which  reference  has  been  made  in  the  section  upon  Strepto- 
coccus pyogenes. 

A  second  important  method,  and  one  that  not  only 
differentiates  the  pneumococcus  from  the  streptococcus,  but 
from  the  common  organisms  of  similar  morphology  that 
infect  the  mouth,  is  the  inulin-serum  water  fermentation 
test  of  Hiss.*  In  using  this  medium,  Ruedigerf  found  it 
best  prepared  as  follows:  Dissolve  5  gm.  of  NaCl,  20 
gm.  of  Witte's  peptone,  and  20  gm.  of  pure  inulin  in  1000 
c.c.  of  distilled  water.  Add  20  c.c.  of  a  5  per  cent,  solu- 
tion of  pure  litmus,  and  tube,  putting  2  c.c.  of  the  mixture 
into  each  tube,  and  sterilize  in  the  autoclave.  After  steril- 
ization add  (with  a  sterile  pipet)  2  c.c.  of  sterile,  heated 
ascitic  fluid,  or,  preferably,  heated  beef-serum,  to  each  tube, 
and  incubate  twenty-four  hours  before  using.  Great  care 
must  be  taken  not  to  use  ascitic  fluid  that  contains  ferment- 
able carbohydrates.  Each  lot  must  be  tested  with  some 
strongly  fermentative  bacterium,  and  the  absence  of  ferment- 
able carbohydrates  proved.  Ruediger  prefers  this  prepa- 
ration to  the  original  solution  of  Hiss  because  he  found  that 
some  pneumococci  would  not  grow  on  the  latter.  Fermenta- 
tion of  the  inulin  is  regarded  as  characteristic  of  the  pneumo- 
coccus. 

The  pneumococcus  produces  red  colonies  upon  litmus- 
inulin-agar  plates,  which  makes  their  use  desirable  when 
pneumococci  are  to  be  isolated  from  saliva,  throat  secretions, 
or  other  material  in  which  similar  appearing  organisms 
are  apt  to  occur.  Ruediger  found  no  other  mouth  bacteria 
that  produced  red  colonies  on  these  plates. 

Immunity. — Pneumonia  is  peculiar  in  that  recovery  is 
followed  by  immunity  of  such  brief  duration  as  to  permit 
the  occurrence  of  frequent  relapses;  and  it  is  well  known 
that  many  cases  show  a  subsequent  predisposition  to  fresh 
attacks  of  the  disease. 

Immune  Serum. — G.  and  F.  Klemperert  have  shown 
that  the  serum  of  rabbits  immunized  against  the  pneumo- 
coccus protects  animals  infected  with  virulent  cultures. 
When  applied  to  human  medicine,  the  serum  failed  to 
do  good. 

The  treatment  of  pneumonia  by  the  injection  of  blood- 

*  "Jour.  Amer.  Med.  Assoc.,"  1906,  vol.  XLVII,  p.  1171. 

t  "Jour,  of  Exp.  Med.,"  1905,  vol.  vi,  p.  317. 

J  "Berliner  klin.  Wochenschrift,"  1891,  Nos.  34  and  35. 


506  Pneumonia 

serum  from  convalescent  patients,  tried  by  Hughes  and 
Carteras,*  has  been  abandoned  as  useless  and  dangerous. 

More  recent  antipneumococcic  sera  have  been  experi- 
mentally investigated  by  De  Renzi,f  Washbourn,  t  and 
Pane.§ 

Washbourn  prepared  an  antipneumococcus  serum  that 
protected  rabbits  against  ten  times  the  fatal  dose  of  live 
pneumococci,  in  doses  of  0.3  c.c.  In  general,  the  lines  upon 
which  he  operated  were  those  of  Behring,  Marmorek's  work 
with  the  streptococcus  furnishing  most  of  the  details. 
Two  cases  of  human  pneumonia  seem  to  have  derived  some 
benefit  from  large  doses  of  this  serum.  The  sera  of  Pane 
and  De  Renzi  were  not  so  powerful  as  those  of  Washbourn, 
requiring  about  i  c.c.  to  protect  a  rabbit. 

McFarland  and  Lincoln  ||  succeeded  in  immunizing  a 
horse  against  large  doses  of  a  virulent  culture  of  the  pneumo- 
coccus,  and  obtained  a  serum  of  which  0.5  to  0.25  c.c.  pro- 
tected rabbits  from  many  times  the  fatal  dose. 

The  experiments  by  Passler**  showed  some  gain  over  the 
earlier  work. 

The  antipneumococcic  sera  thus  far  produced  have  given 
disappointing  results  in  clinical  application. 

A  leukocytic  extract  prepared  by  Hiss  and  Zinsser|t 
from  an  aleuronat  exudation  in  the  rabbit's  pleura  has  led 
to  results  sufficiently  encouraging  in  the  treatment  of 
pneumonia  in  man  to  warrant  further  investigation  along 
similar  lines. 

RosenowJI  found  that  pneumococci  suspended  in  sodium 
chlorid  solutions  autolyse  rapidly.  By  means  of  this  au- 
tolysis  it  is  possible  to  separate,  at  least  to  a  large  degree,  the 
toxic  from  the  antigenic  parts  of  the  pneumococcus,  as  the 
toxic  part  goes  into  solution.  The  injection  of  the  non- 
toxic  and,  as  it  appears,  antigenic  portion — autolyzed  pneu- 
mococci— causes  a  marked  increase  in  the  immunity  curve 

*  "Therapeutic  Gazette,"  Oct.  15,  1892. 
t  "II  Policlinico,"  Oct.  31,  1896,  Supplement. 
I  "Brit.  Med.  Jour.,"  Feb.  27,  1897,  p.  510. 

§  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  May  29,  1897,  xxi,  17  and  18, 
p.  664. 

||  "Jour.  Amer.  Med.  Assoc.,"  Dec.  16,  1899,  p.  1534. 
**  "Deutsches  Archiv  fur  klin.  Med.,"  Bd.  LXXXII,  Nos.  3,  4,  1905; 
"Jour.  Amer.  Med.  Assoc.,"  May  13,  1905,  p.  1538. 
ft  "Jour.  Med.  Research,"  1908,  xix,  323. 
tJ  "Jour.  Amer.  Med.  Assoc.,"  June  10,  LIV,  No.  24,  p.  1943- 


Sanitation  507 

as  measured  by  the  specific  increase  in  pneumococcus 
opsonin.  The  injection  of  such  autolyzed  pneumococci  into 
25  patients  with  lobar  pneumonia  seemed  to  have  a  marked 
beneficial  effect. 

Sanitation. — Pneumonia  is  undoubtedly  a  contagious 
disease.  Exactly  how  infection  takes  place  is  not  known, 
but  seeing  that  the  infectious  agent  is  in  the  respiratory  tract, 
from  which  it  is  easily  discharged  into  the  atmosphere  during 
cough,  etc.,  and  the  facility  with  which  it  can  then  be  inhaled 
by  those  nearby,  it  seems  justifiable  to  conclude  that  the 
primary  entrance  of  the  organism  into  the  body  is  through 
the  respiratory  tract.  Wood*  has  shown  that  "  the  organ- 
isms in  the  sputum  do  hot  remain  long  in  suspension  and  die 
off  rapidly  under  the  action  of  light  and  desiccation.  In 
sunlight  or  diffuse  daylight  the  bacteria  in  such  powder  die 
within  an  hour,  and  in  about  four  hours  if  kept  in  the  dark. 
The  danger  of  infection  from  powdered  sputum  may,  there- 
fore, be  avoided  by  ample  illumination  and  ventilation  of  the 
sick-room  in  order  to  destroy  or  dilute  the  bacteria,  and  by 
the  avoidance  of  dry  sweeping  or  dusting.  Articles  which 
may  be  contaminated  and  which  cannot  be  cleaned  by  cloths 
dampened  in  a  suitable  disinfectant  should  be  removed  from 
the  patient's  vicinty. 

PNEUMOCOCCUS    (FRIEDLANDER) — BACTERIUM  PNEUMONIA 
(ZOPFJ) — BACILLUS  CAPSULATUS  Mucosus  (FASCHINGJ). 

General  Characteristics. — An  encapsulated,  non-motile,  non-flag- 
ellated, non-sporogenous,  non-liquefying,  aerobic  and  optionally  anaer- 
obic, non-chromogenic,  aerogenic  and  pathogenic  bacillus,  staining  by 
ordinary  methods,  but  not  by  Gram's  method. 

This  organism  was  discovered  by  Friedlander§  in  1883  in 
the  pulmonary  exudate  from  a  case  of  croupous  pneumo- 
nia, and,  being  thought  by  its  discoverer  to  be  the  cause  of 
that  disease,  was  called  the  pneumococcus,  and  later  the 
pneumobacillus.  The  grounds  upon  which  the  specificity  of 
the  organism  was  supposed  to  depend  were  soon  found  to 
be  insufficient,  and  the  organism  of  Friedlander  is  at  pres- 
ent looked  upon  as  one  whose  presence  in  the  lung  is,  in 

*  "Jour.  Exp.  Med.,"  Aug.  25,  1905,  vn,  No.  5,  p.  624. 

f  "Spaltpilze,"  1885,  p.  66. 

t  "Centralbl.  f.  Bakt.,"  etc.,  xn,  1892,  p.  304. 

§  "Fortshritte  der  Medizin,"  1883,  22,  715. 


508 


Pneumonia 


most  cases,  unimportant,  though  it  is  sometimes  asso- 
ciated with  and  is  probably  the  cause  of  a  special  form 
of  lobular  pneumonia,  which,  according  to  Stiihlern,*  is 
clinically  atypical  and  usually  fatal.  Frankel  points  out  that 
Friedlander's  error  in  supposing  his  organism  to  be  the  chief 
parasite  in  pneumonia  depended  upon  the  fact  that  his  studies 
were  made  by  the  plate  method,  which  permitted  the  dis- 
covery of  this  bacillus  to  be  made  more  easily  than  that  of 
the  slowly  growing  and  more  delicate  pneumococcus.  In 
the  light  of  present  knowledge  Friedlander's  bacillus  must  be 


Fig.  164. — Bacterium  pneumonicum  (modified  after  Migula). 

looked  upon  as  the  type  of  a  group  of  organisms  varying  among 
themselves  in  many  minor  particulars. 

Distribution. — The  organism  is  sometimes  found  in 
normal  saliva;  it  is  a  common  parasite  of  the  respiratory 
apparatus,  not  infrequently  occurs  in  purulent  accumula- 
tions, is  occasionally  found  in  feces,  and  sometimes  occurs 
under  external  saprophytic  conditions.  Thus  it  is  probably 
identical  with  that  described  as  the  "  capsulated  canal-water 
bacillus  "  by  Mori,f  and  may  be  identical  with  or  at  least 
belong  to  the  same  group  in  which  we  find  Bacillus  aerogenes 
capsulatus. 

*"Centralbl.  f.  Bakt.,"  etc.  (Originale),  Bd.  xxxvi,  No.  4,  p.  493, 
July  21,  1904. 

t  "Zeitschnft  fur  Hygiene,"  iv,  1888,  p.  53. 


Cultivation 


509 


Morphology. — Though  usually  dis- 
tinctly bacillary  in  form,  the  organism 
is  of  variable  length  and  when  paired 
sometimes  bears  a  close  resemblance 
to  the  pneumococcus  of  Frankel  and 
Weichselbaum  It  measures  0.5  to 
1.5  ^  in  breadth  and  0.6  to  0.5  [A  in 
length.  It  frequently  occurs  in 
chains  of  four  or  more  elements  and 
occasionally  appears  elongated.  It  is 
these  variations  in  form  that  have 
led  to  the  description  of  the  organism 
by  different  writers  as  a  coccus,  a 
bacterium,  and  a  bacillus.  It  is  com- 
monly surrounded  by  a  distinct  trans- 
parent capsule,  hence  its  name  "  cap- 
sule bacillus"  and  Bacillus  capsulatus 
mucosus.  The  organism  is  non- 
motile,  has  no  spores,  and  no  flagella. 
It  stains  well  with  the  ordinary  anilin 
dyes,  but  does  not  retain  the  color 
when  stained  by  Gram's  method. 

Cultivation. — Colonies. — If  pneu- 
monic exudate  be  mixed  with  gelatin 
and  poured  upon  plates,  small  white 
spheric  colonies  appear  at  the  end  of 
twenty-four  hours,  and  spread  out 
upon  the  surface  of  the  gelatin  to 
form  whitish  masses  of  a  considerable 
size.  Under  the  microscope  these 
colonies  appear  irregular  in  outline 
and  somewhat  granular. 

Bouillon. — There  is  nothing  char- 
acteristic about  the  bouillon  cultures 
of  Friedlander's  bacillus.  The  me- 
dium is  diffusely  clouded.  A  pellicle 
usually  forms  on  the  surface  and  a 
viscid  sediment  soon  accumulates. 

Gelatin  Puncture. — When  a  colony 
is  transferred  to  a  gelatin  puncture 
culture,  a  luxuriant  growth  occurs. 
Upon  the  surface  a  somewhat  ele- 
vated, rounded  white  mass  is  formed, 


Fig.  165. — Friedlan- 
der's pneumobacillus ; 
gelatin  stab  culture, 
showing  the  typical 
nail-head  appearance 
and  the  formation  of 
gas  bubbles,  not  always 
present  (Curtis). 


510  Pneumonia 

and  in  the  track  of  the  wire  innumerable  little  colonies 
spring  up  and  become  confluent,  so  that  a  ' '  nail-growth ' ' 
results.  No  liquefaction  of  the  gelatin  occurs.  Gas  bubbles 
not  infrequently  appear  in  the  wire  track.  The  cultures 
sometimes  become  brown  in  color  when  old. 

Agar-agar. — Upon  the  surface  of  agar-agar  at  ordinary 
temperatures  a  luxuriant  white  or  brownish-yellow,  smeary, 
viscid,  circumscribed  growth  occurs. 

Blood-serum. — The  blood-serum  growth  is  similar  to  that 
upon  agar. 

Potato. — Upon  potato  the  growth  is  luxuriant,  quickly 
covering  the  entire  surface  with  a  thick  yellowish -white 
layer,  which  sometimes  contains  bubbles  of  gas. 

Milk  is  not  coagulated  as  a  rule.     Litmus  milk  is  reddened. 
Vital  Resistance. — The  bacillus  grows  at  a  temperature 
as  low  as  16°  C.,  and,  according  to  Sternberg,  has  a  thermal 
death-point  of  56°  C. 

Metabolic  Products. — Friedlander's  bacillus  ferments 
nearly  all  the  sugars,  with  the  evolution  of  much  gas.  It 
generates  alcohol,  acetic  and  other  acids,  and  both  CO2  and 
H.  According  to  the  best  authorities  the  organism  does  not 
form  indol.  There  is,  however,  some  difference  of  opinion 
upon  the  subject. 

Perkins*  divides  the  organisms  of  this  group  into  three 
chief  types  according  to  their  reactions  toward  carbohy- 
drates : 

I.  Bacillus  aerogenes  type  ferment  all  carbohydrates, 

with  the  formation  of  gas. 

II.  Bacillus  pneumoniae  (Friedlander)  type  ferment  all 
carbohydrates  except  lactose,  with  formation  of 
gas. 

III.  Bacillus  lactis  aerogenes  type  ferment  all  carbo- 
hydrates except  saccharose,  with  formation  of 
gas. 

Pathogenesis. — Friedlander  found  considerable  difficulty 
in  producing  pathogenic  changes  by  the  injection  of  his 
bacillus  into  the  lower  animals.  Rabbits  and  guinea-pigs 
were  immune  to  its  action,  and  the  only  important  patho- 
genic effects  that  Friedlander  observed  occurred  in  mice, 
into  whose  lungs  and  pleura  he  injected  the  cultures,  with 
resulting  inflammatory  lesions. 

That  Friedlander's  bacillus  may  be  the  cause  of  true  lobar 
*  ''Jour,  of  Infect.  Dis.,"  1904,  i,  No.  2,  p.  241. 


Pathogenesis  511 

pneumonia  there  can  be  no  room  for  doubt  after  the  demon- 
strations of  Lamon  and  Meltzer,*  who  found  that  its  experi- 
mental introduction  into  the  bronchi  of  dogs  was  followed  by 
true  lobar  pneumonia.  The  lesions  in  these  dogs,  like  those 
in  human  beings,  were  paler  in  color,  the  lung  tissue  less 
friable,  and  the  exudate  more  viscid  than  those  caused  by 
the  pneumococcus. 

Pneumonia  in  man,  caused  by  Bacillus  mucosus  capsulatus, 
is  atypical  clinically,  very  severe,  and  often  fatal. 

Curryt  found  Friedlander's  bacillus  in  association  with  the 
pneumococcus  in  acute  lobar  pneumonia;  in  association  with 
the  diphtheria  bacillus  in  otitis  media  associated  with  croup- 
ous  pneumonia;  and  in  the  throat  in  diphtheria.  In  pure 
culture  it  was  obtained  from  vegetations  upon  the  valves  of 
the  heart  in  a  case  of  acute  endocarditis  with  gangrene  of  the 
lung;  from  the  middle  ear,  in  a  case  of  fracture  of  the  skull 
with  otitis  media ;  and  from  the  throat  in  a  case  of  tonsillitis. 
Zinsser  has  twice  cultivated  Friedlander's  bacillus  from  in- 
flamed tonsils  in  children. 

AbelJ  cultivated  it  from  the  discharges  of  fetid  ozena, 
and  supposed  it  to  be  the  specific  cause. 

Occasionally  Friedlander's  bacillus  bears  an  important 
relationship  to  lobular  or  catarrhal  pneumonia,  an  interesting 
case  having  been  studied  by  Smith.  §  The  histologic  changes 
in  the  lung  were  remarkable  in  that  the  "  alveolar  spaces 
of  the  consolidated  areas  were  dilated  and  for  the  most 
part  filled  with  the  capsule  bacilli."  In  some  alveoli  there 
seemed  to  be  pure  cultures  of  the  bacilli;  others  contained 
red  and  white  blood-corpuscles;  in  some  there  was  a  little 
fibrin.  The  bacillus  obtained  from  this  case,  when  injected 
into  the  peritoneal  cavity  of  guinea-pigs,  produced  death 
in  eleven  hours.  The  peritoneal  cavity  after  death  con- 
tained a  large  amount  of  thick,  slimy  fluid;  the  intestines 
were  injected  and  showed  a  thin  fibrinous  exudate  upon  the 
surface;  the  spleen  was  enlarged  and  softened,  and  the 
adrenals  much  reddened.  Cover-glass  preparations  from 
the  heart,  blood,  spleen,  and  peritoneal  cavity  showed  large 
numbers  of  the  capsule  bacilli. 

*  "Jour.  Exp.  Med.,"  1912,  xv,  133. 

f  "Jour.  Boston  Soc.  of  Med.  Sci.,"  March,  1898,  vol.  n.  No.  8,  p.  137. 

t  "Zeitschrift  fur  Hygiene,"  xxi. 

§  "Jour.  Boston  Soc.  of  Med.  Sci.,"  May,  1898,  vol.  n,  No.  10,  p.  174. 


512  Pneumonia 

Howard*  has  also  called  attention  to  the  importance  of 
this  bacillus  in  connection  with  numerous  acute  and  chronic 
infectious  processes,  among  which  may  be  mentioned 
croupous  pneumonia,  suppuration  of  the  antrum  of  High- 
more  and  frontal  sinuses,  endometritis,  perirenal  abscesses, 
and  peritonitis. 

Virulence. — The  virulence  of  the  organism  seems  to  vary 
under  different  conditions.  It  is  sometimes  harmless  for 
the  experiment  animals,  but  when  injected  into  mice  and 
guinea-pigs  usually  produces  local  inflammatory  lesions,  and 
sometimes  invasion  of  the  circulation  and  death  from  sepsis. 

CATARRHAL  PNEUMONIA  OR  BRONCHOPNEUMONIA. 

This  form  of  pulmonary  inflammation  occurs  in  local 
areas,  commonly  situated  about  the  distribution  of  a  bron- 
chiole. It  cannot  be  said  to  have  a  specific  micro-organism, 
as  almost  any  irritating  foreign  matter  accidentally  inhaled 
may  cause  it.  The  majority  of  the  cases,  however,  are 
infectious  in  nature  and  result  from  the  inspiration,  from 
higher  parts  of  the  respiratory  apparatus,  of  the  staphylo- 
cocci  and  streptococci  of  suppuration,  Friedlander's  bacillus, 
the  bacillus  of  influenza,  and  other  well-known  organisms. 

TUBERCULAR  PNEUMONIA. 

The  progress  of  pulmonary  tuberculosis  is  at  times  so 
rapid  that  the  tubercle  bacilli  are  distributed  with  the 
softened  infectious  matter  throughout  the  entire  lung  or  to 
large  parts  of  it,  and  a  distinct  pneumonic  inflammation 
occurs.  Such  a  pneumonia  may  be  caused  by  the  tubercle 
bacillus,  but  more  frequently  depends  upon  accompanying 
staphylococci,  streptococci,  tetragenococci,  pneumococci, 
pneumobacilli,  and  other  organisms  accidentally  present  in 
a  lung  in  which  ulceration  and  cavity  formation  are  ad- 
vanced. 

PLAGUE  PNEUMONIA. 

The  pneumonic  form  of  plague  is  characterized  by  con- 
solidation of  the  lung  histologically  and  anatomically,  indis- 
tinguishable from  pneumococcal  and  other  extensive  pul- 
monary infections. 

*  "Phila.  Med.  Jour.,"  Feb.  19,  1898,  vol.  i,  No.  8,  p.  336. 


Mixed  Pneumonias  513 


MIXED   PNEUMONIAS. 

It  frequently  happens  that  pneumonia  occurs  in  the  course 
of  influenza  or  shortly  after  convalescence  from  it.  In  these 
cases  a  mixed  infection  by  the  influenza  bacilli  and  pneu- 
mococci  is  commonly  found.  Sometimes  pneumococci  and 
staphylococci  simultaneously  affect  the  lung,  purulent  pneu- 
monia with  abscess  formation  being  the  conspicuous  feature. 
Almost  any  combination  of  the  described  bacteria  may  occur 
in  the  lungs,  producing  varying  inflammatory  conditions, 
so  that  it  must  be  left  for  the  student  to  work  out  what  the 
particular  characteristics  of  each  may  be. 

Among  the  mixed  forms  of  pneumonia  may  be  mentioned 
those  called  by  Klemperer  and  Levy  "  complicating  pneu- 
monias," occurring  in  the  course  of  typhoid  fever,  etc. 

33 


CHAPTER  XVI. 

INFLUENZA. 
BACILLUS  INFLUENZA  (R.  PFEIFFER). 

General  Characteristics. — A  minute,  non-motile,  non-flagellated, 
non-sporogenous,  non-liquefying,  non-chromogenic,  aerobic,  pathogenic 
bacillus,  staining  by  the  ordinary  methods,  b.ut  not  by  Gram's  method, 
and  susceptible  of  artificial  cultivation,  chiefly  through  the  addition 
of  hemoglobin  to  the  culture-media. 

Notwithstanding  the  number  of  examinations  conducted 
to  determine  the  cause  of  influenza,  it  was  not  until  1892, 
after  the  great  epidemic,  that  Pfeiffer*  found,  in  the  blood 
and  purulent  bronchial  discharges,  a  bacillus  that  con- 
formed, in  large  part,  to  the  requirements  of  specificity. 

Morphology. — The  bacilli  are  very  small,  having  about 
the  same  diameter  as  the  bacillus  of  mouse  septicemia,  but 
only  half  its  length  (0.2  by  0.5  fi).  They  are  usually  soli- 
tary, but  may  be  united  in  chains  of  three  or  four. 

They  are  non-motile,  have  no  flagella,  and,  so  far  as  is 
known,  do  not  form  spores. 

Staining. — They  stain  rather  poorly  except  with  such 
concentrated  and  penetrating  stains  as  carbol-fuchsin  and 
Loffler's  alkaline  methylene-blue,  and  even  with  these  more 
deeply  at  the  ends  than  in  the  middle,  so  that  they  appear 
not  a  little  like  diplococci.  They  do  not  stain  by  Gram's 
method. 

Canon  f  recommends  a  rather  complicated  method  for  the 
demonstration  of  the  bacilli  in  the  blood.  The  blood  is 
spread  upon  clean  cover-glasses  in  the  usual  way,  thoroughly 
dried,  and  then  fixed  by  immersion  in  absolute  alcohol  for 
five  minutes.  The  best  stain  is  Czenzynke's: 

Concentrated  aqueous  solution  of  methylene-blue 40 

0.5  per  cent,  solution  of  eosin  in  70  per  cent,  alcohol.  .20 
Distilled  water 40 

.*  "  Deutsche  med.  Wochenschrift,"  1892, 2 ;  "Zeitschrift  fur  Hygiene," 
13- 

t  "Centralbl.  f.  Bakt.,"  etc.,  Bd.  xiv,  p.  860. 


Isolation 


The  cover-glasses  are  immersed  in  the  solution,  and  kept 
in  the  incubator  for  from  three  to  six  hours,  after  which  they 
are  washed  in  water,  dried,  and  mounted  in  Canada  balsam. 
By  this  method  the  erythrocytes  are  stained  red,  the  leuko- 
cytes blue;  and  the  bacilli,  also  blue,  appear  as  short  rods 
or  as  dumb-bells. 

Large  numbers  of  bacilli  may  be  present,  though  some- 
times only  a  few  can  be  found  after  prolonged  search,  as  they 
are  prone  to  occur  in  widely  scattered  but  dense  clusters. 
They  are  frequently  inclosed  within  the  leukocytes.  It  is 


Fig.  166. — Bacillus  of  influenza.     Smear  from  sputum.     (After  Heim.) 


scarcely  necessary  to  pursue  so  tedious  a  staining  method  for 
demonstrating  the  bacilli,  for  they  stain  well  enough  for  recog- 
nition by  ordinary  methods. 

Isolation. — The  influenza  bacillus  grows  poorly  upon 
artificial  culture-media,  and  is  not  easy  to  isolate,  because 
the  associated  bacteria  tend  to  outgrow  it.  When  isolated 
it  is  difficult  to  keep,  as  it  soon  dies  in  unnatural  environ- 
ment. 

Pfeiffer  found  that  the  organism  grew  when  he  spread  pus 
from  the  bronchial  secretions  upon  serum-agar.  Subcul- 
tures made  from  the  original  colonies  did  not  "take."  It 
therefore  seemed  as  though  it  might  depend  upon  the  absence 
of  some  ingredient  that  the  bronchial  secretions  contained. 


Influenza 


By  a  series  of  experiments  he  was  able  to  make  the  organism 
grow  when  he  transferred  it  to  agar-agar,  the  surface  of  which 
was  coated  with  a  film  of  blood  taken,  with  precautions  as  to 
sterility,  from  the  finger-tip.  Later  it  was  found  that  the 
addition  of  hemoglobin  to  the  culture-medium  was  equally 
efficacious.  By  the  use  of  such  blood-smeared  agar  and  gly- 
cerin-agar  the  organism  can  now  be  successfully  cultivated. 
The  isolation  is  best  achieved  through  the  use  of  bronchial 
secretions,  carefully  washed  in  sterile  water  or  salt  solution 
to  remove  contaminating  organisms  from  the  mouth. 


Fig.    167. — Bacillus  of  influenza;  colonies  on  blood  agar-agar.     Low 
magnifying  power  (Pfeiffer). 


Cultivation. — Upon  blood-spread  glycerin  agar-agar, 
after  twenty-four  hours  in  the  incubator,  minute  colorless, 
transparent,  dewdrop-like  colonies  may  be  seen  along  the  line 
of  inoculation.  They  look  like  condensed  moisture,  and  Kita- 
sato  makes  a  special  point  of  the  fact  that  they  never  become 
confluent.  The  colonies  may  at  times  be  so  small  as  to 
require  a  lens  for  their  detection. 

No  growth  takes  place  at  room  temperature.  The  organ- 
isms die  quickly  and  must  be  transplanted  every  three  or  four 
days  if  they  are  to  be  kept  alive. 


Immunity  517 

The  organism  is  aerobic  and  scarcely  grows  at  all  where  the 
supply  of  oxygen  is  not  free. 

In  bouillon  a  scant  development  occurs,  small  whitish 
particles  appearing  upon  the  surface,  subsequently  sinking 
to  the  bottom  and  causing  a  "wooly"  deposit  there.  The 
bacillus  grows  more  luxuriantly  upon  culture-media  contain- 
ing hemoglobin  or  blood,  and  can  be  transferred  from  culture 
to  culture  many  times  before  losing  vitality. 

Vital  Resistance. — Its  resisting  powers  are  very  re- 
stricted, as  it  speedily  succumbs  to  drying,  and  is  certainly 
killed  by  an  exposure  to  a  temperature  of  60°  C.  for  five 
minutes.  It  will  not  grow  at  any  temperature  below  28°  C. 

Specificity. — From  the  fact  that  the  bacillus  is  found 
chiefly  in  cases  of  influenza,  that  it  is  present  as  long  as  the 
purulent  secretions  of  the  disease  last,  and  then  disappears, 
and  that  Pfeiffer  was  able  to  demonstrate  its  presence  in  all 
cases  of  uncomplicated  influenza,  it  seems  that  his  conclu- 
sion that  the  bacillus  is  specific  is  justifiable.  It  is  also 
found  in  the  secondary  morbid  processes  following  influenza, 
such  as  pneumonia,  endocarditis,  middle-ear  disease,  menin- 
gitis, etc.  Horder*  has  cultivated  it  from  the  valvular  vege- 
tation of  2  cases  of  endocarditis  following  influenza. 

Davis  f  found  the  influenza  bacillus  in  the  respiratory 
passage  of  a  large  number  of  patients  suffering  from  whoop- 
ing-cough. 

Pathogenesis. — The  bacillus  is  pathogenic  for  very  few 
of  the  laboratory  animals,  the  guinea-pig  being  susceptible 
of  fatal  infection.  The  dose  required  to  cause  death  of  a 
guinea-pig  varies  considerably. 

Pfeiffer  and  BeckJ  produced  what  may  have  been  influenza 
in  monkeys  by  rubbing  their  nasal  mucous  membranes  with 
pure  cultures. 

Immunity. — As  influenza  is  a  disease  that  commonly  re- 
lapses, and  from  which  one  rarely  seems  to  acquire  protection 
against  future  attacks,  there  must  be  scarcely  any  immunity 
induced  through  ordinary  infection.  Moreover,  the  organism 
once  finding  its  way  into  the  body  seems  to  remain  almost  in- 
definitely, especially  when,  as  in  pulmonary  tuberculosis, 
there  is  already  present  an  abnormal  condition  furnishing 
discharges  or  exudates  in  which  it  can  thrive. 

*  "Path.  Soc.  of  London,"  "Brit.  Med.  Jour.,"  April  22,  1905. 
t  "Jour.  Infectious  Diseases,"  in,  1906,  i. 
J"  Deutsche  med.  Wochenschrift,"  1893,  xxi. 


Influenza 


In  the  immunization  experiments  of  Delius  and  Kolle* 
one-twentieth  of  a  twenty-four-hour-old  culture  was  fatal  in 
twenty-four  hours.  They  found  that  the  toxicity  of  the  cul- 
ture does  not  depend  upon  a  soluble  toxin,  but  upon  an  intra- 
cellular  toxin.  The  outcome  of  the  researches,  which  were 
made  most  painstakingly,  was  total  failure  to  produce  experi- 
mental immunity. 

Increasing  doses  of  the  cultures,  injected  into  the  perito- 
neal cavity,  enabled  the  animals  to  resist  more  than  a  fatal 


Fig.  1 68. — Bacillus  of  influenza;  cover-glass  preparation  of  sputum 
from  a  case  of  influenza,  showing  the  bacilli  in  leukocytes.  Highly 
magnified  (Pfeiffer). 

dose,  but  never  enabled  them  to  maintain  vitality  when  large 
doses  of  living  cultures  were  administered.  This  observation 
is  in  exact  harmony  with  the  familiar  clinical  observation 
that,  instead  of  an  individual  remaining  immune  after  an 
attack  of  influenza,  he  is  quite  as  susceptible  as  before. 

A.  Catanni,  Jr.,f  trephined  rabbits  and  injected  influenza 
toxin  into  their  brains,  at  the  same  time  trephining  control 
animals,  into  some  of  whose  brains  he  injected  water.  The 
animals  receiving  0.5  to  i  mg.  of  the  living  culture  died  in 

*  "Zeitschrift  fur  Hygiene,"  etc.,  Bd.  xxiv,  1897,  Heft  2. 
t  Ibid.,  Bd.  xxm,  1896. 


The  Pseudo-Influenza  Bacillus  519 

twenty-four  hours  with  all  the  nervous  symptoms  of  the  dis- 
ease, dyspnea,  paralysis  beginning  in  the  posterior  extremities 
and  extending  over  the  whole  body,  clonic  convulsions,  stiff- 
ness of  the  neck,  etc.  Control  animals  injected  in  the  same 
manner  with  water,  and  with  a  variety  of  other  pathogenic 
bacteria  never  manifested  similar  symptoms.  The  viru- 
lence of  the  bacillus  increased  rapidly  when  transplanted 
from  brain  to  brain. 

Diagnosis  of  Influenza. — Wynekoop*  employs  for  diag- 
nosticating influenza  and  isolating  the  bacillus,  a  culture 
outfit  similar  to  that  used  for  diphtheria  diagnosis,  except 
that  the  serum  contains  more  hemoglobin.  The  swab  is 
used  to  secure  secretions  from  the  pharynx  and  tonsils,  and 
from  the  bronchial  secretions  of  patients  with  influenza,  then 
rubbed  over  the  blood-serum.  In  many  such  cultures 
minute  colonies  corresponding  to  those  of  the  influenza 
bacillus  were  found.  Those  most  isolated  were  picked  up 
with  a  wire  and  transplanted  to  bouillon,  from  which  fresh 
blood-serum  was  inoculated  and  pure  cultures  secured. 

Carbol-fuchsin  was  found  most  useful  for  staining  the 
bacilli.  Wynekoop  observed  that  influenza  and  diphtheria 
bacilli  sometimes  coexist  in  the  throat,  and  that  influenza 
bacilli  are  present  in  the  sore  eyes  of  those  in  the  midst 
of  household  epidemics  of  influenza. 

THE  PSEUDO-INFLUENZA  BACILLUS. 

Pfeiffer  f  has  also  described  a  pseudo-influenza  bacillus — a 
small,  non-motile,  non-flagellated,  non-sporogenous,  Gram- 
negative  bacillus — that  he  found  in  certain  cases  of  broncho- 
pneumonia  in  children.  It  differed  from  the  influenza  bacillus 
by  a  slightly  greater  size,  a  tendency  to  grow  in  chains,  and 
to  undergo  involution.  Martha  Wollsteinf  believes  that 
they  are  influenza  bacilli. 

*  ''Bureau  and  Division  Reports,"  Department  of  Health,  city  of 
Chicago,  Jan.,  1899. 

t  " Zeitschrif t  fur  Hygiene,"  etc.,  1892,  xm. 
t  "Jour.  Exp.  Med.,"  1906,  vm. 


CHAPTER   XVII. 
MALTA  OR  MEDITERRANEAN  FEVER. 

MICROCOCCUS  MEWTENSIS  (BRUCE);  BACILLUS  MEUTENSIS 

(BABES). 

General  Characteristics. — A  non-motile,  non-flagellate,  non-spo- 
rogenous,  non-chromogenic,  'non-liquefying,  pathogenic  coccus,  staining 
by  the  ordinary  methods,  but  not  by  Gram's  method;  characterized 
by  remarkably  slow  growth  and  by  pathogenic  action  upon  monkeys. 

In  1877,  while  working  in  Malta,  Bruce*  succeeded  in 
finding  in  every  fatal  case  of  Malta  fever  a  micrococcus 
which  could  be  isolated  in  pure  cultures  from  the  spleen, 
liver,  and  kidney,  which  grew  readily  on  artificial  media,  and 
which,  when  injected  into  monkeys,  produced  the  disease. 

Morphology. — Micrococcus  melitensis,  as  Bruce  called  it, 
is  a  round  or  slightly  oval  organism  measuring  about  0.3  ^ 
in  diameter.  It  is  usually  single,  sometimes  in  pairs,  but 
never  in  chains.  When  viewed  in  the  hanging  drop  it  is 
said  to  exhibit  active  "molecular"  movements,  but  is  not 
motile  and  has  no  flagella.  Babes  f  declares  it  to  be  a  bacillus. 

Staining. — It  stains  well  with  aqueous  solutions  of  the 
anilin  dyes,  but  not  by  Gram's  method. 

Thermal  Death  Point. — This  has  been  fixed  by  Dalton 
and  Eyre  J  at  57.5°  C. 

Cultivation. — The  best  medium  for  its  cultivation  is 
said  to  be  ordinary  agar-agar.  After  inoculating,  by  a  punc- 
ture, from  the  spleen  of  a  fatal  case  of  Malta  fever,  the  tubes 
should  be  kept  at  37°  C.  The  growth  first  appears  after 
several  days,  in  the  form  of  minute  pearly  white  spots  scat- 
tered around  the  point  of  puncture  and  along  the  needle 
path.  After  some  weeks  the  colonies  grow  larger  and  join 
to  form  a  rosette-like  aggregation,  while  the  needle  tract  be- 
comes a  solid  rod  of  yellowish-brown  color.  After  a  lapse  of 
months  the  growth  still  remains  restricted  to  the  same  area 
and  its  color  deepens  to  buff. 

*  ''Practitioner,"  xxxiv,  p.  161. 

t  Kolle  and  Wassermann  "Die  Pathogenic  Mikroorganisms,"  m,  p. 
443- 

t  "Jour,  of  Hygiene,"  iv.,  1904,  p.  157. 

520 


Bacteriologic  Diagnosis 


When  the  sloping  surface  of  inoculated  agar-agar  is  ex- 
amined by  transmitted  light,  the  appearance  of  the  colonies 
is  somewhat  different.  At  the  end  of  nine  or  ten  days,  if 
kept  at  37°  C.,  some  of  the  colonies  have  a  diameter  of  2  to  3 
mm.  They  are  round  in  form,  have  an  even  contour,  are 
slightly  raised  above  the  surface  of  the  agar-agar,  and  are 
smooth  and  shining  in  appearance.  On  examining  the 
colonies  by  transmitted  light,  the  center  of  each  is  seen  to 
be  yellowish,  while  the  periphery  is  bluish-white  in  color. 
The  same  colonies  by  reflected  light  appear  milky  white  in 
color.  Colonies  on  the  surface  of  the  agar-agar  are  found 
to  be  no  larger  than  hemp-seed  after  a  couple  of  months 
of  cultivation. 

When  kept  at  25°  C.,  no  colonies  become  visible  to  the 


*   - 


Fig.  169.  —  Micrococcus  melitensis. 


naked  eye  before  the  seventh  day;  at  37°  C.,  before  the 
third  or  fourth  day. 

The  growth  in  gelatin  takes  place  at  room  temperature 
with  great  slowness,  first  appearing  in  about  a  month, 
and  no  liquefaction  of  the  medium  occurs. 

No  growth  takes  place  on  boiled  potato. 

Plate  cultures  are  not  adapted  to  the  study  of  the  organ- 
ism because  of  its  extreme  slowness  of  growth. 

Bacteriologic  Diagnosis.  —  The  specific  agglutinative 
effect  of  the  serum  can  be  made  use  of  for  the  purpose 


522  Malta  or  Mediterranean  Fever 

of  diagnosis.     This  has  been  studied  by  Wright,*  Birt  and 
Lamb,f  and  later  by  Bassett-Smith.J 

All  of  the  observers  have  shown  that  the  agglutinative 
reaction  takes  place  both  with  living  and  dead  cultures  of 
the  Micrococcus  melitensis,  but  that  to  make  the  diagnosis 
dilutions  of  serum  equal  to  about  i :  30,  never  greater  than 
i :  50,  must  be  used.  Birt  and  Lamb  also  arrive  at  certain 
conclusions  regarding  the  prognosis  based  upon  a  study  of 
the  agglutinative  phenomena.  Their  conclusions  are : 

1.  Prognosis  is  unfavorable  if  the  agglutinating  reaction  is  per- 

sistently low. 

2.  Also  if  the  agglutinating  reaction  rapidly  fall  from  a  high  figure 

to  almost  zero. 

3.  A  persistently  high  and  rising  agglutinating  reaction  sustained 

into  convalescence  is  favorable. 

4.  A  long  illness  may  be  anticipated  if  the  agglutination  figure,  at 

first  high,  decreases  considerably. 

The  agglutination  reaction  appears  early,  and  is  available 
by  the  end  of  the  first  week,  and  persists  often  for  years 
after  convalescence. 

The  organisms  may  sometimes  be  cultivated  from  the 
blood  taken  from  a  vein,  but  are  more  certainly  to  be  secured 
by  splenic  puncture. 

Pathogenesis. — The  micro-organism  is  not  pathogenic 
for  mice,  guinea-pigs,  or  rabbits,  but  is  fatal  to  monkeys 
when  agar-agar  cultures  suspended  in  water  are  injected 
beneath  the  skin. 

The  micro-organism  usually  seems  to  be  absent  from  the 
circulating  blood,  though  Hughes  has  cultivated  it  from  the 
heart's  blood  of  a  dead  monkey. 

Bruce  not  only  succeeded  in  securing  the  micro-organism 
from  the  cadavers  of  Malta  fever,  but  has  also  obtained  it 
during  life  by  splenic  puncture. 

Accidental  inoculation  with  micrococcus  melitensis,  as  by 
the  prick  of  a  hypodermic  needle,  is  almost  invariably  fol- 
lowed by  an  attack  of  the  disease.  Six  cases  of  this  kind 
occurred  in  connection  with  bacteriologic  work  on  Malta 
fever  at  Netley  and  two  additional  at  the  Royal  Naval 
Hospital  at  Haslar  and  in  the  Philippines.  § 

Treatment — The   treatment  of   Mediterranean  fever  by 

*  "Lancet,"  1897,  March  6;  "Brit.  Med.  Jour.,"  1897,  May  15. 

f  Ibid.,  1899,  n,  p.  701. 

J  "British  Med.  Jour.,"  1902,  n,  p.  861. 

§  See  Wright  and  Windsor,  "Jour,  of  Hygiene,"  n,  1902,  p.  413. 


Goats'  Milk  and  Malta  Fever  523 

means  of  bacterio-vaccines  has  been  attempted  with  what 
seems  to  be  glittering  results  by  Bassett-Smith.* 

The  report  of  "  British  Government  Commission  for  the 
Investigation  of  Mediterranean  Fever,"  published  by  the 
Royal  Society,  April,  1907,  has  greatly  elucidated  our  knowl- 
edge of  the  pathogen y  of  the  disease  by  showing  that  the 
Micrococcus  melitensis  leaves  the  body  of  the  patient  in  the 
urine  and  in  the  milk.  It  has  not  been  found  in  the  saliva, 
sweat,  breath,  or  feces.  The  discovery  of  the  organism  in 
the  milk  suggested  that  it  might  be  through  milk  that  the 
disease  organisms  were  disseminated,  and  an  investigation  of 
the  goats  at  Malta,  where  the  disease  is  most  prevalent,  and 
their  milk  most  generally  used,  showed  that  a  large  per- 
centage of  the  animals  were  infected  with  the  specific  cocci. 
The  commission  has,  therefore,  concluded  that  it  is  by 
goats'  milk  that  the  disease  is  commonly  disseminated, 
though  they  point  out  that  fly-transmission  is  also  possible. 
In  the  Colonial  Office  Report  on  Malta  in  1907  it  was  shown 
that  over  40  per  cent,  of  the  goats  of  Malta  gave  the  serum 
reaction,  showing  that  they  had  had  the  disease,  while  10  per 
cent,  of  them  were  actually  secreting  the  cocci  in  their  milk. 
The  authorities  permit  no  milk  to  be  used  in  the  garrison 
unless  it  is  boiled,  and  notice  that  by  this  simple  measure 
the  incidence  of  the  disease,  which  was  9.6  in  1905,  had 
fallen  to  2  in  the  corresponding  month  of  1906.  In  Report 
VII.  of  the  Mediterranean  Fever  Commission  (1906-07)  we 
read: 

"The  epidemiologists  are  led  to  believe  that  quite  70  per  cent,  of 
the  cases  are  due  to  the  ingestion  of  goat's  milk."  In  their  opinion 
ordinary  contact  with  the  sick,  conveyance  of  infection  by  biting  insects, 
house  flies,  dust,  drain  emanations,  food  (other  than  milk),  and  water, 
play  a  very  subordinate  part,  if  any,  in  setting  up  Mediterranean  fever 
in  man.  The  excellent  results  following  the  preventive  measures  di- 
rected against  goat's  milk  in  barracks  and  hospitals  also  point  to  goat's 
milk  as  being  the  chief  factor.  Among  the  soldiers  this  resulted  in  a 
diminution  of  about  90  per  cent. 

"For  example,  in  the  second  half  of  1905  there  were  363  cases  of 
Mediterranean  fever,  whereas  in  the  corresponding  part  of  1906  there 
were  only  35  cases.  Among  the  sailors  there  was  also  as  marked  a  fall 
in  the  number  of  cases.  The  Naval  Hospital  had  a  bad  reputation,  as 
about  one-third  of  the  cases  of  fever  occurring  in  the  fleet  at  Malta 
could  be  traced  to  residence  in  this  hospital,  either  as  patients  suffering 
from  other  diseases  or  among  the  nursing  staff.  The  goats  supplying 
the  hospital  were  found  to  be  infected,  and  since  their  milk  was  absolutely 
forbidden,  not  a  single  case  of  Malta  fever  has  occurred  in  or  been  traced 
to  residence  in  this  hospital." 

*  "Journal  of  Hygiene,"  vn,  1907,  p.  115. 


CHAPTER   XVIII. 

MALARIA. 

MALARIA,  or  paludism,  has  been  known  since  the  days  of 
ancient  medicine,  and  has  always  been  regarded  as  the 
typical  miasmatic  disease.  Its  name,  mala  aria,  means 
"  bad  air,"  and  is  Italian  derived  from  the  Latin,  mains  and 
aer,  coming  from  the  Greek  dyp,  air,  from  dew,  to  blow. 
The  other  name,  paludism,  from  the  Latin  pains,  a  "  marsh," 
refers  the  disease  to  the  bad  air  coming  from  marshes. 

It  is  a  disease  of  extremely  wide  geographic  distribution, 
and  since  the  supposed  requirement,  marshy  ground,  is 
found  in  nearly  all  countries,  and  the  disease  is  particularly 
prevalent  in  the  marshy  districts  of  those  countries  in  which 
it  occurs,  the  connection  between  the  marshes  and  the  disease 
seemed  clear.  Indeed,  the  two  are  intimately  connected, 
but  not  in  the  original  sense,  as  will  be  shown  below. 

Both  hemispheres,  all  of  the  continents,  and  most  of  the 
islands  of  the  sea  suffer  more  or  less  from  malaria,  and  in 
many  places,  especially  in  the  tropics,  it  is  so  pestilential  as 
to  make  the  country  uninhabitable.  Probably  no  better 
idea  of  the  wide  distribution  and  severity  of  the  disease  can 
be  obtained  than  by  reference  to  Davidson's  "  Geographical 
Pathology."  * 

The  disease  assumes  the  form  of  a  fever  of  intermittent  or 
remittent  type,  characterized  by  certain  peculiar  paroxysms. 
When  typical,  as  in  well-marked  intermittent  fever,  these  are 
ushered  in  by  depression,  headache,  and  chilly  sensations, 
which  are  soon  followed  by  pronounced  rigors  in  which  the 
patient  shivers  violently,  his  teeth  chattering.  The  tem- 
perature soon  begins  to  rise  and  attains  a  height  of  102°,  104°, 
or  even  106°  F.,  according  to  the  severity  of  the  case.  As 
the  temperature  rises  the  sense  of  chilliness  disappears  and 
gives  place  to  burning  sensations.  The  skin  is  flushed,  hot, 
and  dry.  After  a  period  varying  in  length  the  skin  begins 
to  break  out  into  perspiration,  which  is  soon  profuse,  the 

*  D.  Appleton  &  Co.,  New  York,  1892. 
524 


Malaria  525 

fever  and  headache  disappear  and  the  patient  commonly 
sinks  into  a  refreshing  sleep.  The  frequency  of  the  paroxysms 
varies  with  the  type  of  the  disease,  which,  in  its  turn,  can  be 
referred  to  the  kind  of  infection  by  which  it  is  caused.  The 
paroxysms  exhaust  the  patient  and  incapacitate  him  and 
may  eventually  prove  fatal,  though  in  by  far  the  greater 
number  of  cases  the  disease  gradually  expends  itself  and  a 
partial  or  complete  recovery  ensues.  Some  cases,  known 
as  pernicious,  are  rapidly  fatal,  others  develop  into  a 
chronic  cachexia,  with  profound  anemia  and  complete  in- 
capacitation  for  physical  or  mental  effort.  The  discovery 
of  Peruvian  or  Jesuits'  bark,  and  its  introduction  into  Europe 
by  the  Countess  del  Cinchon,  the  wife  of  the  Viceroy  of  Peru, 
about  1639,  marked  an  important  epoch  in  the  study  of 
malarial  fever.  The  isolation  of  its  alkaloids,  quinin  and 
cinchona,  begun  in  1810  by  Gomez  and  perfected  in  1820 
by  Pelletier  and  Coventou,  a  second  great  epoch.  But  the 
most  important  epoch  began  in  1880,  when  Charles  Louis 
Alphonse  Laveran,*  a  French  physician  engaged  in  the  study 
of  malarial  fever  in  Algeria,  announced  the  discovery  of  a 
parasite,  to  which  he  gave  the  name  Plasmodium  malariae, 
in  the  blood  of  patients  suffering  from  the  disease.  His  ob- 
servations were  immediately  confirmed,  Biitschli  recognizing 
the  parasitic  nature  of  the  bodies  observed.  For  the  discov- 
ery he  was  awarded  the  Breant  prize. 

Laveran,  however,  threw  no  light  upon  the  source  of  in- 
fection, and  malaria  continued  to  be  described  as  a  mias- 
matic disease. 

It  was,  however,  recognized  that  there  were  different 
types  of  parasites  corresponding  to  the  different  clinical 
forms  of  the  disease,  and  Golgif  succeeded  in  correlating 
the  various  appearances  of  the  parasites  so  as  to  express 
their  life  cycles.  But  in  spite  of  the  interesting  and  im- 
portant work  of  Golgi,  Celli,  Bignami  and  Marchiafava,  and 
many  others  no  progress  was  made  in  accounting  for  the 
entrance  of  the  parasites  into  the  human  body. 

This  problem  had  long  interested  Sir  Patrick  Manson, 
who  had  devised  a  theory  which,  though  wrong  in  de- 
tail, proved  in  the  end  to  open  the  door  to  the  next  im- 
portant discovery.  Finding  that  the  malarial  parasites 
could  not  be  shown  to  leave  the  body  in  any  of  its  elimina- 

*  "Acad.  d.  Med.,"  Paris,  Nov.  28  and  Dec.  28,  1880. 
t  "R.  Acad.  di  Medicina  di  Torino,"  1885,  xi,  20. 


526  Malaria 

tions,  and  remembering  that  the  same  was  true  of  the  filarial 
worms  and  their  embryos,  Manson  came  to  the  conclusion 
that  they  must  be  taken  out  of  the  blood  by  some  suctorial 
insect.  The  one  naturally  first  considered  was  the  mosquito, 
which  was  known  to  abound  wherever  malaria  prevailed. 
Examining  mosquitoes  that  had  been  permitted  to  distend 
themselves  with  the  blood  containing  the  parasites,  Manson 
found  that  in  the  stomach  of  the  insect  the  peculiar  phenom- 
enon known  as  "  flagellation,"  long  before  observed  by  Lav- 
eran,  took  place  in  the  parasites,  giving  rise  to  long,  slender, 
lashing,  and,  finally,  free-swimming  filaments.  These,  he 
conjectured,  might  be  the  form  in  which  the  parasites  left 
the  mosquito  to  infect  the  swamp  water,  with  which  human 
infection  eventually  was  brought  about.  Here  Manson 
failed,  but  while  he  was  investigating  he  explained  the 
whole  matter  to  Major  Ronald  Ross,  who  was  soon  to  go  to 
India,  and  whom  he  advised  to  make  the  matter  a  subject  for 
study  when  he  arrived  at  his  destination.  Ross*  accepted 
the  opportunity  that  soon  presented  itself,  and,  after  a  most 
painstaking  investigation,  the  details  of  which  are  given  in  a 
paper  which  can  be  found  in  the  International  Medical  An- 
nual, f  1890,  made  the  second  great  discovery  in  the  parasit- 
ology  of  malarial  ever.  He  found  that,  as  Manson  thought, 
the  mosquito  is  the  definitive  host  of  the  parasite,  but  that  the 
matter  is  much  less  simple  than  was  imagined,  for  the  organ- 
isms taken  up  by  the  mosquito  undergo  a  complicated  life 
cycle  requiring  about  a  fortnight  for  completion,  after  which, 
not  the  water  into  which  the  mosquito  might  fall  and  into 
which  its  contained  organisms  might  escape,  but  the  mosquito 
itself  becomes  the  agent  of  infection.  In  other  words,  the 
parasites  taken  up  by  the  mosquito,  after  the  completion  of 
the  necessary  developmental  cycle,  are  returned  by  the  mos- 
quito to  new  human  beings,  who  thus  become  infected.  Thus 
it  was  shown  that  malaria  is  not  a  miasmatic  disease  at  all, 
but  that  it  is  an  infectious  disease  whose  parasites  divide  their 
life  cycle  between  man  and  the  mosquito,  each  becoming 
infected  by  the  other.  The  only  role  of  the  swamp  is  to 
furnish  the  mosquitoes,  and  since  these  are  only  more  numer- 
ous, where  swamps  are  numerous,  but  may  occur  without 
swamps,  the  not  infrequent  occurrence  of  malarial  fevers 
apart  from  swamps  is  also  explained.  Ross  further  discov- 

*  "Indian  Medical  Gazette,"  xxxui,  14,  133,  401,  448. 
t  E.  B.  Treat  &  Co.,  New  York. 


Malarial  Parasites  527 

ered  that  all  mosquitoes  are  not  equally  susceptible  of  infec- 
tion, and,  therefore,  not  all  able  to  spread  the  infection. 
Indeed,  he  so  carefully  studied  the  mosquitoes  as  to  narrow 
the  infectability  and  infectivity  of  mosquitoes  down  to  one 
single  family,  the  Anophelinae,  and  to  one  single  genus, 
Anopheles. 

There  remained,  however,  one  more  important  fact  to  be 
elucidated,  and  one  more  mysterious  body  to  be  accounted 
for,  viz.,  the  "  flagellated  "  body  that  had  misled  Manson. 
This  was  found  by  MacCallum*  to  be  but  the  spermatozoit 
of  the  male  parasite.  While  observing  one  of  the  malarial 
parasites  of  birds — Plasmodium  danliewskyi — he  saw  one  of 
these  "  flagella  "  swimming  away  from  its  parent  parasite,  and 
followed  it  carefully,  moving  the  slide  upon  the  stage  of  the 
microscope.  It,  and  others  of  its  kind,  approached  a  large 
globular  parasite,  to  which  one  effected  an  attachment  and 
into  which  it  entered.  MacCallum  realized  that  he  had 
observed  the  sexual  fertilization  of  the  organism.  Thus 
from  its  time-honored  place  as  the  typical  miasmatic  disease, 
full  of  mystery  and  obscurity,  malarial  fever  suddenly  had 
a  flood  of  light  thrown  upon  it  by  which  every  peculiarity  was 
fully  illuminated. 

In  summarizing  the  knowledge  thus  set  forth  we  find  the 
following  facts : 

1880 — Discovery  of  the  Plasmodium  malarise  by  Laveran. 

1890 — Discovery  of  its  human  developmental  cycle  by 
Golgi. 

1895 — Discovery  of  the  mosquito  cycle  and  mode  of  trans- 
mission by  Ross. 

iSgS: — Discovery  of  the  sexual  fertilization  of  the  parasite 
by  MacCallum. 

The  interest  aroused  by  Laveran's  original  discovery  gave 
a  great  impetus  to  the  study  of  hematology  with  special  refer- 
ence to  parasites,  and  it  soon  became  evident  that  the  plas- 
modium  was  but  one  of  a  group  of  similar  parasites.  Of 
these  we  have  now  become  acquainted  with  the  following: 

Parasite.  Disease.  Host.  Insect  host. 

Plasmodium  Quartan  fever.  Man.  Anopheles,  My- 

malariae.  zorrhynchus, 

Myzomyia. 

Plasmodium  Tertian  fever.  Man.  Anopheles,  My- 

vivax.  zorrhynchus, 

Myzomyia. 

*"Jour.  of  Exper.  Med.,"  1898,  in,  117. 


528 


Malaria 


Parasite.  Disease. 

Plasmodium  fal-     Aestivo  -  autum- 
ciparum  nal  fever. 

Plasmodium 

kochi. 
Plasmodium 

inui. 

Plasmodium 
pitheci. 

Plasmodium 

brazilianum. 
Plasmodium 

cynomolgi. 

Plasmodium 
grassii     (Pro- 
teosoma  gras- 
sii). 

Plasmodium 
danliewskyi 
(Halteridium 
danliewskyi) . 


Host. 


Man. 


Cercopithicus. 

Macacus  (In- 
uus  cyno- 
molgus). 

Orang  -  outang 
(Pithecus  sa- 
tyrus) . 

Brachyrus  cal- 
ores. 

Inuus  cynomol- 
gus  and  Inuus 
nemistrinus. 

Sparrows,     can- 
ary birds,  and 
other    small 
birds. 

Owls,  hawks, 
crows,  and 
other  large 
birds. 


Insect  host. 

Anopheles,  My- 
zorrhynchus, 
Myzomyia. 

Unknown. 

Unknown. 


Unknown. 

Unknown. 
Unknown. 

Culex  pipens. 
Unknown. 


These  micro-organisms  correspond  in  all  essentials.  They 
are  protozoan  parasites  belonging  to  the  sporozoa  and  live 
in  the  blood  (hematozoa)  as  parasites  of  the  red  corpuscles. 
They  all  have  two  life  cycles,  one  which  is  asexual  in  the 
intermediate  warm-blooded  host,  and  one  that  is  sexual  in 
the  definitive  cold-blooded  (insect)  host.  Though  the  inter- 
mediate hosts  vary  and  may  be  birds  or  mammals,  the 
insect  hosts,  so  far  as  known,  are  always  mosquitoes.  The 
mosquitoes  become  infected  by  biting  and  sucking  the 
blood  of  infected  animals;  the  warm-blooded  animals  be- 
come infected  by  being  bitten  by  infected  mosquitoes,  and  so 
on,  in  endless  cycles. 

The  parasites  differ  but  little  in  the  details  of  structure 
and  development,  so  that  the  following  description  may  serve 
as  a  type  for  all : 

From  the  proboscis  of  the  mosquito,  with  its  saliva,  from 
cells  in  the  salivary  glands  where  they  have  been  harbored, 
tiny  elongate  spindles,  measuring  about  1.5  ^  in  length  and 
0.2  fi  in  breadth,  and  know  as  sporozoits,  enter  the  blood  of 
the  individual  bitten.  These  sporozoits  attach  themselves 
to  the  red  blood-corpuscles,  gradually  lose  their  elongate 
form,  and  become  irregularly  spherical.  There  is  some 
difference  of  opinion  as  to  whether  the  little  bodies  are  simply 
upon  the  corpuscles,  as  Koch  believed,  or  in  the  corpuscles, 


Asexual  Life  Cycle 


529 


as  the  majority  of  writers  believe,  but  it  is  an  immaterial 
difference,  for  the  parasite  soon  makes  clear  that  it  is  con- 
suming the  corpuscle.  This  little  body  is  known  as  a 
schizont.  When  stained  with  polychrome  methylene-blue, 
and  examined  under  a  high  power  of  the  microscope,  it  ap- 


Fig.  170. — Plasmodium  falciparum.     Ookinetes  in  the  stomach  of  An- 
opheles (Grassi). 

pears  as  a  little  ring  with  a  dark  chromatin  dot  upon  one 
side.  It  grows  steadily,  feeding  upon  the  hemoglobin,  which 
seems  to  be  chemically  transformed  into  fine  or  coarse  gran- 
ules of  a  bacillary  or  rounded  form,  presumably  melanin. 
In  a  length  of  time  that  varies — 
twenty-four  to  forty-eight  hours 
(Plasmodium  falciparum),  forty- 
eight  hours  (Plasmodium  vivax), 
seventy-two  hours  (Plasmodium 
malariae) — the  schizonts  mature, 
becoming  nearly  as  large  or  quite 
as  large  as  the  corpuscles.  The 
pigment  granules  now  collect  at 
the  center  and  the  substance  of 
the  parasite  divides  into  a  group 
of  equal-sized  merozoits,  com- 
monly known  as  spores.  Of  these  cipafum.  Transverse  section 
there  are  usually  eight  in  the  °J  the  stomach  of  Anopheles, 
,,  f  *r*t  ,.  showing  the  ookmetes  of  the 

meroblasts  of  Plasmodium    ma-      parasite   in  various  stages  of 
lariae,  from  fifteen  to  twenty-five     development  attached  to  the 
in  those  of  Plasmodium  vivax,     outer  surf  ace  (Grassi) . 
and  from    eight   to   twenty-five 

in  Plasmodium  falciparum.  As  the  spores  become  fully 
formed  and  ready  to  separate,  the  paroxysm  of  the  disease 
begins.  It  ends  as  the  spores  are  freed  and  enter  new 
corpuscles  to  begin  the  cycle  over  again.  After  a  good  many 
34 


Fig.  171. — Plasmodium  fal- 


22 


Fig.  172. — Developmental  cycle  of  plasmodium  vivax,  the  tertian 
malarial  parasite.  Figures  i  to  17  are  magnified  1200  diameters;  18  to 
27,  only  600  diameters:  i,  Sporozoit;  2,  penetration  of  a  sporozoit  into  a 
red  blood-corpuscle;  3  and  4,  schizont  developing  in  the  red  blood-cor- 
puscles; 5  and  6,  nuclear  division  of  the  schizont;  7,  free  merozoits;  8 
(following  the  arrows  to  the  left  to  3),  merozoits  entering  red  blood-cor- 
puscles, and  multiplying  by  schizogony  3  to  7 ;  after  longer  continuance 
of  the  disease  the  sexual  forms  arise;  ga  to  12 a,  macrogametocytes;  9b 


Sexual  Life  Cycle  531 

paroxysms  have  occurred  it  may  be  observed  that  not  all  of 
the  schizonts  change  to  meroblasts  and  form  spores.  Some 
remain  large  spheroidal  bodies  or,  as  in  Plasmodium  falci- 
parum,  assume  a  peculiar  crescentic  form  and  remain  un- 
changed in  the  blood.  These  are  the  sexual  parasites.  The 
female  is  usually  the  larger  and  is  known  as  the  makrogame- 
tocyte,  the  male,  the  smaller,  the  micro  gametocyte.  These 
are  the  bodies  which  when  removed  by  the  mosquito 
lay  the  foundation  of  its  infection.  When  they  are  with- 
drawn for  microscopic  examination  or  exposed  to  the  in- 
testinal juices  of  the  mosquito,  the  microgametocyte  be- 
comes tumultuous,  its  granules  are  observed  to  be  in  a  state 
of  active  cytoplasmic  streaming,  and  suddenly  there  burst 
forth  long  slender  filaments,  the  micro  gametes  or  sporozoits. 
These  correspond  with  the  flagella  of  Laveran  and  others, 
and  are  the  same  bodies  that  Manson  thought  might  be 
the  form  in  which  the  parasite  leaves  the  insect's  body. 
The  microgametes  lash  vigorously  for  a  time,  then,  breaking 
loose,  swim  away,  and,  as  MacCallum  observed,  conjugate 
with  macrogametes  sexually  perfect  cells  formed  from  the 
macrogametocytes,  thus  fertilizing  them.  As  the  result  of 
this  fertilization  a  zygote  or  ookinete  is  formed.  It  assumes 
a  somewhat  elongate  pointed  form  and  attaches  itself 
to  the  wall  of  the  mosquito's  stomach.  In  the  course 
of  time  it  penetrates  and  appears  upon  the  outside,  pro- 
jecting into  the  body  cavity.  It  grows  larger  and  rounder, 
divides  into  several  segments,  and  eventually  forms  an 
oocyst  with  many  small  cells,  which  break  up  into  myriads 
of  tiny  elongate  fusiform  bodies,  the  sporozoits.  These, 
in  the  course  of  time,  seem  to  find  their  way  to  the  sali- 

to  i2b,  microgametocytes  still  in  the  circulatory  blood  of  man.  If  the 
macrogametocytes  (i2a)  are  not  taken  into  the  alimentary  canal  of  the 
mosquito,  they  multiply  parthenogenetically  (i2a,  I3C  to  lye)  and  the 
resulting  merozoits  (lye)  become  schizonts  (3  to  7).  The  figures  below 
the  dotted  line  represent  what  takes  place  in  the  alimentary  canal  of 
anopheles  (13  to  17);  i3b  and  i^b  the  formation  of  microgametocytes; 
i3a  and  i3b,  maturation  of  the  macrogametes;  isb,  a  microgamete;i  6, 
fertilization;  17,  ookinete;  18,  ookinete  on  the  wall  of  the  mosquito's 
stomach;  19,  penetration  of  the  gastric  epithelium  by  the  ookinetes;  20 
to  25,  stages  of  sporogenesis  on  the  outer  wall  of  the  mosquito's  stomach; 
26,  migration  of  the  sporozoits  to  the  salivary  glands  of  the  mosquito;  27, 
salivary  gland  with  sporozoits  in  the  epithelial  cells,  and  escape  of  the 
sporozoits  from  the  salivary  glands  through  the  insect's  proboscis  at 
the  time  a  human  host  is  bitten;  i,  free  sporozoit  from  the  mosquito's 
saliva  in  the  human  blood;  2,  penetration  of  the  sporozoit  into  a  red 
blood-corpuscle,  beginning  the  human  cycle  again  (Liihe). 


532  Malaria 

vary  glands,  entering  into  the  epithelial  cells  and  taking 
radial  positions  about  the  nuclei,  where  they  remain  for  a 
time.  Later,  they  leave  the  cells  with  the  saliva,  and  when 
the  mosquito  again  bites,  enter  the  warm-blooded  host  to 
infect  it,  if  of  the  appropriate  species. 

The  whole  cycle  in  the  mosquito  varies,  according  to  the 
external  temperature,  from  ten  days  to  a  fortnight.  The 
mosquito  may  remain  alive  for  more  than  one  hundred  days, 
and  must  bite  frequently  to  satisfy  its  needs.  It  remains 
infective  so  long  as  the  sporozoits  remain  in  the  saliva,  which 
is  usually  as  long  as  the  insect  is  alive.  Here  it  may  be 
remarked  that  as  it  is  only  the  female  mosquitoes  that  bite, 
it  is  only  by  them  that  the  infection  can  be  spread.  It  is 
an  interesting  question,  not  yet  solved,  whether  any  of  the 
sporozoits  entering  into  the  mosquito's  ovaries  can  infect 
its  eggs  so  that  a  new  generation  of  mosquitoes  may  be  born 
infective.  The  longer  the  human  infection  persists,  the 
greater  the  number  of  gametocytes  formed,  until  sometimes, 
especially  in  aestivo-autumnal  malaria,  no  schizonts  are  any 
longer  found,  though  the  blood  contains  large  numbers  of 
gametocytes.  In  such  cases  the  gametocytes,  especially  the 
crescents  of  sestivo-autumnal  fever,  but  sometimes  also  those 
of  tertian  and  quartan  fever  undergo  regressive  schizogony  in 
the  patient's  blood,  and  without  fertilization  suddenly  break 
up  into  spores  which  enter  the  red  blood-corpuscles  and 
occasion  a  relapse  of  the  infection  that  had  apparently  spent 
itself. 

THE  HUMAN  MALARIAL  PARASITES. 

There  are  three  known  forms  of  human  malarial  parasites: 
Plasmodium  malariae,  Plasmodium  vivax,  and  Plasmodium 
falciparum. 

I.  Plasmodium  Malariae  (Laveran,*  1880). — This  is  the 
smallest  of  the  human  malarial  parasites.  Its  occurrence  is 
relatively  infrequent,  as  is  that  of  the  quartan  fever  that 
it  occasions.  The  schizogonic  period  is  seventy-two  hours 
long,  and  as  each  is  completed,  a  paroxysm  of  the  disease 
occurs. 

The  parasite,  in  the  red  blood-corpuscle,  first  appears  as 
a  tiny  ring,  at  one  side  of  which  there  is  a  chromatin  dot. 
At  this  time  the  organism  cannot  be  differentiated  from 

*  "Acad.  de  Med.,"  Nov.  23,  Dec.  28,  1880. 


The  Human  Malarial  Parasites 


533 


Plasmodium  vivax.  At  the  end  of  twenty-four  hours  the 
organism  seems  to  extend  itself  more  or  less  linearly,  and 
sometimes  appears  as  a  long  drawn  band  which  crosses 
the  substance  of  the  unchanged  corpuscle.  In  another 
twenty-four  hours  the  breadth  of  the  parasite  is  two  or 
three  times  as  great,  and  it  has  become  pigmented.  The 
corpuscle  itself  is  still  unchanged.  In  the  last  twenty- 
four  hours  the  parasite  enlarges,  becomes  more  or  less 
quadrilateral,  finally  rounds  up,  shows  depressions  upon 
the  surface  corresponding  to  the  divisions  into  which  it .  is 
to  segment,  the  pigment  gathers  at  the  center,  and  the 
substance  undergoes  cleavage  resulting  in  the  formation  of 
from  six  to  fourteen,  but  usually  eight,  spores.  It  is  to  be 


Fig.  173. — Parasite  of  quartan  malarial  fever:  a,  b,  c,  d,  Enlarging 
intracellular  parasites;  e,  },  g,  h,  segmentating  parasites  forming  a  dis- 
tinct rosette  from  which  the  spores  separate;  i,  macrogametocyte;  ;, 
microgametocyte;  k,  flagellum. 

noticed  that  it  is  not  until  a  few  hours  before  segmentation 
that  the  parasite  becomes  as  large  as  the  corpuscle,  and  that 
the  corpuscle  is  never  enlarged  or  bleached  by  the  presence 
of  the  parasite.  The  meroblasts  form  regular  rosettes,  or 
"daisy-heads,"  within  the  corpuscles. 

In  single  infections  the  parasites  are  all  of  the  same  age 
and  all  mature  at  the  same  time,  so  that  in  any  examination 
of  the  blood  they  will  all  appear  uniform.  It  is,  however, 
sometimes  true  that  the  patient  may  have  been  infected  one 
day  by  one  mosquito  bite,  and  again  infected  the  next  day 
or  the  third  day  by  a  second  mosquito  bite,  so  that  his  blood 
contains  two  crops  of  the  microparasites,  arriving  at  maturity 
at  different  times.  This  perplexes  the  clinician  through  the 
variety  of  parasitic  forms  in  the  blood  and  the  abnormal  fre- 
quency of  the  paroxysms. 


534  Malaria 

The  gametocytes  of  the  parasite  remain  for  some  time  in 
the  red  corpuscles  without  division,  but,  finally,  become  free 
spherical  bodies.  Two  sizes  can  be  made  out,  one  a  little 
larger,  the  macrogametocyte  or  female,  the  other,  the 
microgametocyte  or  male.  Bach  has  protoplasm,  with  a  tend- 
ency to  take  a  blue-gray  color  and  appear  uniformly  granu- 
lar, except  that  at  some  part  of  the  periphery  of  each  there  is 
a  circular  or  semicircular  area  that  is  free  from  granules. 
This  area  is  larger  in  the  microgametocyte. 


Fig.  174-  Fig-  J75- 

Figs.  174,  175. — Gametocytes  of  plasmodium  malariae:  85,  The  macro- 
gametocyte;  86,  the  microgametocyte  (Kolle  and  Wassermann). 

II.  Plasmodium  Vivax  (Grassland  Feletti,*  1890).— This 
is  the  most  common  of  the  malarial  parasites  of  man,  and 
occasions  the  "  benign  "  tertian  fever.  It  is  a  large  parasite, 
the  full-grown  schizont  (meroblast) ,  ready  to  form  merozoits, 
and  the  gametocytes  all  exceeding  the  size  of  the  red  blood- 
corpuscles.  It  matures  in  forty-eight  hours,  but  not  with 
mathematic  precision.  In  single  infections  the  greater 
number  of  the  parasites  are  of  the  same  age  and  present  the 
same  appearance,  but  various  shapes  and  ages  may  be  found 
together.  In  double  infections,  with  paroxysms  every  day, 
parasites  of  different  ages  may  be  found. 

The  youngest  form  in  which  the  parasite  can  be  observed 
is  that  of  a  tiny  ring  in  a  red  blood-corpuscle.  The  periphery 
of  this  ring  (when  the  blood  is  stained  with  polychrome 
methylene-blue)  is  outlined  with  blue,  at  one  side  there  is 

*  "  Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1890,  vn,  396;  1891,  x,  449, 
481,  517. 


PLATE  I 


o 


• 


w 


o 


10 


11 


12 


PLATE  II 


13 


mm 


15 


-•. 


16 


17 


18 


19 


o 


o 


20 


21 


22 


*.*- 
> 


m 


. 


25 


26 


The  Human  Malarial  Parasites 


535 


a  distinct  blue  dot,  and  the  center  appears  colorless  and  like 
a  vacuole.  The  dot  is  usually  on  the  side  of  the  vacuole  that 
has  the  thinner  protoplasmic  outline.  The  smallest  such 
rings  usually  have  a  diameter  equal  to  about  ^  the  diameter 
of  the  blood-corpuscle.  The  tiny  ring-form,  or,  as  it  might 
better  be  called,  the  "seal-ring  form,"  continues  until  the 
schizont  becomes  half  the  diameter  of  the  blood-corpuscle, 
when  its  protoplasm  has  begun  to  increase  so  rapidly  that 
the  vacuole  no  longer  appears  to  be  so  conspicuous.  The 
organism  also  becomes  irregular  in  shape  and  is  actively 


Fig.  176. — Parasite  of  tertian  malarial  fever:  a,  b,  c,  d,  e,  f,  g,  Grow- 
ing pigmented  parasite  in  the  red  blood-corpuscles;  h,  spores  formed 
by  segmentation  of  the  parasite — no  rosette  is  formed,  but  concentric 
rings  of  the  cytoplasm  divide;  i,  macrogametocyte;  ;,  microgametocyte 
with  flagella. 

ameboid,  its  protoplasm  streaming  this  way  and  that  when 
examined  in  fresh  blood.  At  this  time  it  may  be  noticed 
that  the  infected  blood-corpuscle  is  increasing  in  volume, 
sometimes  becoming  twice  the  normal  size,  and  also  becoming 
pale  in  color.  It  seems  also  as  though  the  disk  shape  of  the 
corpuscle  was  lost,  and  it  had  become  swollen  into  a  more 
spherical — sometimes  irregular — form.  The  parasite,  which 
may  still  show  a  relic  of  its  original  ring- form,  now  shows  plen- 
tifully throughout  its  protoplasm  exceedingly  fine  granules  of 
yellow-brown  pigment.  When  from  thirty-six  to  forty  hours 
old,  all  trace  of  the  "  seal-ring  "  form  disappears,  the  ame- 


536 


Malaria 


boid  action  becomes  less  marked,  and  the  parasites  (now 
three-quarters  the  size  of  the  enlarged  pale  and  misshapen 
corpuscles  in  which  they  are  contained)  appear  as  irregular, 
ragged,  protoplasmic  bodies  filled  with  fine  pigment  granules. 
In  about  forty-five  hours  they  completely  fill  the  enlarged 
corpuscles,  and  begin  to  gather  their  protoplasm  into  rounded 
formations  in  which  the  pigment  is  no  longer  distributed, 
but  occurs  in  irregular  stripes  or  gathers  together  into  a 
rounded  clump.  In  a  couple  of  hours  the  blood-corpuscle 
has  disappeared  and  the  rounded  parasite,  larger  than 
normal  red  corpuscles,  with  a  tabulated  surface,  and  with  its 
pigment  granules  collected  to  form  one  or  two  rounded 


Q& 


Fig.  177.  Fig.  178. 

Figs.   177,  178. — Gametocytes  of  plasmodium  vivax:  87,  The  micro- 
gametocyte;  88,  the  macrogametocyte  (Kolle  and  Wassermann). 

masses,  is  seen  to  have  reached  the  stage  of  the  meroblast. 
This  does  not  form  the  rosette  or  "  daisy-head  "  shown  by 
the  quartan  parasite,  but  might  better  be  compared  to  a 
mulberry,  and  eventuates  in  the  formation  of  from  fifteen 
to  twenty-five  small,  rounded  or  ovoid,  pale,  unpigmented 
bodies,  the  merozoits  or  spores.  These  become  freed  from 
the  pigment  and  attached  to  new  red  corpuscles,  in  which 
they  are  easily  recognized  as  the  "  tiny  rings  "  that  begin  the 
schizogonic  cycle.  The  gametocytes  of  the  tertian  parasite, 
the  "  free  spheres,"  as  they  are  sometimes  called,  are  large, 
rounded  or  slightly  ovoid  bodies,  with  a  uniformly  dull 
bluish-gray  or  grayish-green  protoplasm,  in  the  interior  of 
which  there  is  always  a  circular  or  semicircular  area  periph- 
erally or  centrally  situated,  and  colorless.  Except  in  this 


The  Human  Malarial  Parasites  537 

area  the  pigment  is  distributed  throughout  the  parasite. 
The  larger  or  macrogametocyte,  the  female  parasite,  measures 
10  to  14  H  in  diameter.  It  has  a  greenish  or  grayish-green 
or  almost  colorless  protoplasm,  containing  an  oval  or  bean- 
shaped  colorless  area  almost  half  as  large  as  the  organism 
itself.  Yellowish-brown  pigment  in  short  broad  rods  is  spar- 
ingly scattered  throughout  the  substance  elsewhere. 

The  microgametocyte  or  male  form  is  approximately  the 
size  of  a  red  blood-corpuscle — 8  to  9  ft  in  diameter.  It 
stains  more  deeply  than  its  mate  and  contains  more  and 
coarser  pigment  granules. 

III.  Plasmodium  Falciparum  (Blanchard,  1897). — This 
is  the  parasite  of  estivo-autumnal  or  malignant  tertian 


Fig.  179.  —  Parasite  of  estivo-autumnal  fever:  a,  b,  c,  Ring-like  and 
cross-like  hyaline  forms;  d,  e,  pigmented  forms;  /,  g,  segmentary  forms; 
h,  i,  j,  crescents. 


malarial  fever.  It  is  a  very  small  parasite,  whose  occur- 
rence, even  multiple  occurrence,  in  the  corpuscles  does  not 
change  their  size  or  shape.  It  does,  however,  quickly  change 
the  appearance  of  the  corpuscles,  which  become  polychro- 
matophilic,  and  frequently  show  numerous  small  dots  —  the 
granulations  of  Schuffner  —  in  the  corpuscular  substance. 

The  first  appearance  of  the  schizont  is  in  the  form  of  tiny 
rings,  which  appear  to  lie  upon  rather  than  in  the  corpuscles, 
and  are  first  seen  at  the  edges.  The  rings  are  outlined  by 
extremely  fine  lines  and  sometimes  seem  to  be  incompletely 
closed,  so  that  they  are  like  horseshoes  rather  than  circles. 
They  increase  to  several  times  the  original  size  without  losing 
the  ring  shape,  and  are  variously  known  as  "middle-sized 
rings  "  and  "  large  rings."  They  are  with  difficulty  differ- 
entiated from  the  "  tiny  rings  "  of  the  tertian  parasite.  As 


538  Malaria 

the  "  large  ring  "  stage  is  reached  the  parasites  begin  to 
disappear  from  the  peripheral  blood  to  complete  their  growth 
and  undergo  meroblast  formation  in  the  capillaries  of  the 
spleen,  the  brain,  and  the  bone-marrow.  Here  the  full- 
grown  parasites — meroblasts — appear  as  irregular  disks, 
resembling  those  of  the  quartan  parasite,  but  smaller  in  size. 
The  pigment  is  gathered  toward  the  center  in  a  little  mass, 
and  eight  to  twenty-five  merozoits  are  formed  in  a  morula 
or  mulberry-like  mass  similar  to  those  of  the  tertian  para- 
site. Two  or  three  parasites  to  the  corpuscle  are  frequent. 
They  are  actively  ameboid,  do  not  mature  simultaneously, 
and  hence  there  are  no  regularly  occurring  paroxysms.  The 


Fig.  1 80.  Fig.  181. 

Figs.  1 80,  181. — Gametocytes  of  plasmodium  falciparum:  91,  The  mi- 
crogametocyte;  92,  the  macrogametocyte  (Kolle  and  Wassermann). 


duration  of  the  asexual  cycle  is  from  twenty-four  to  forty- 
eight  hours. 

The  gametocytes  are  striking  and  characteristic  ovoid  and 
crescentic  bodies — crescents — i  J  times  the  diameter  of  a  red 
blood-corpuscle  in  length,  and  about  half  the  diameter  of  the 
corpuscle  in  breadth.  The  ends  color  more  intensely  with 
methylene-blue  than  the  middle  portion,  and  the  bacillary 
pigment  granules  are  collected  toward  the  centers.  The 
longer  and  more  slender  crescents  are  usually  bent,  and  the 
relic  of  the  corpuscle  in  which  they  have  formed  can  often 
be  seen  forming  a  line  connecting  the  ends  on  the  concave 
side.  These  are  the  microgametocytes  or  male  elements. 
The  macrogametocytes  are  broader,  not  curved,  and  some- 
times are  ovoidal  or  prolate  spheroidal  in  shape.  The  pig- 


Pathogenesis  539 

ment  granules  are  more  widely  scattered  throughout  the  sub- 
stance. The  crescents  are  most  numerous  after  the  fever 
has  lasted  for  some  time  or  in  recurrences  of  the  fever. 

The  fever  in  this  form  of  malarial  infection  may  be  inter- 
mittent with  daily — quotidian — paroxysms,  or  with  irregu- 
lar paroxysms,  or  the  fever  may  be  remittent.  The  infec- 
tion is  sometimes  mild,  but  may  be  so  severe  as  to  be  rapidly 
fatal.  In  such  cases  the  number  of  parasites  is  enormous, 
the  cerebral  capillaries  become  filled  with  them,  and  coma 
quickly  comes  on  and  is  soon  followed  by  death.  Such 
cases  are  described  as  "  congestive  chills  "  or  "  algid  " 
cases. 

Cultivation  of  the  Parasites. — The  parasites  have  not 
been  successfully  cultivated,  though  they  have  been  kept 
alive  for  some  time  in  blood,  prevented  from  coagulation,  by 
Bass. 

Animal  Inoculation. — The  human  malarial  parasites  can- 
not be  successfully  transmitted  by  experimental  inoculation 
to  any  of  the  lower  animals. 

Human  Inoculation. — The  blood  of  one  human  being  con- 
taining schizonts,  when  experimentally  introduced  into  an- 
other human  being  in  doses  of  i  to  1.5  c.c.  transmits  the  dis- 
ease. When  thus  transmitted,  an  incubation  period  of 
from  seven  to  fourteen  days  intervenes  before  the  disease, 
which  is  of  the  same  type  as  that  from  which  the  blood  was 
taken,  makes  its  appearance. 

Pathogenesis. — The  pathogenic  effects  wrought  by  the 
malarial  parasite  are  imperfectly  understood.  The  syn- 
chrony of  the  segmentation  of  the  parasite  and  the  occur- 
rence of  the  paroxysms  seems  to  indicate  that  a  toxic  sub- 
stance saturates  and  disturbs  the  economy  at  that  time. 
Whether  it  be  an  endotoxin  liberated  by  the  dividing  parasite 
is  not,  however,  known. 

The  anemia  that  follows  infection  can  be  referred  to  the 
destruction  of  the  red  blood-corpuscles  by  the  parasites  which 
feed  upon  them  and  transform  the  hemoglobin  into  melanin 
(?).  When  great  numbers  of  the  parasites  are  present  the 
destruction  is  enormous,  and  the  number  of  corpuscles  and 
the  quantity  of  hemoglobin  in  the  blood  sink  far  below  the 
normal.  Leukopenia  instead  of  leukocytosis  is  the  rule,  and 
while  the  leukocytes  have  an  appetite  for  the  spores  of  the 
parasites  and  often  phagocyte  and  destroy  them,  their  activ- 


54°  Malaria 

ity  is  not  sufficiently  rapid  or  universal  to  check  their  rapid 
increase. 

The  melanin  granules  set  free  during  sporulation  are  also 
taken  up  by  the  leukocytes  and  endothelial  cells,  the  latter 
becoming  deeply  pigmented  at  times. 

The  spleen  enlarges  as  the  disease  continues  until  it  forms 
the  "  ague-cake."  The  enlargement  may  cause  the  organ 
to  weigh  7  to  10  pounds.  It  appears  to  result  from  hyper- 
trophy. The  tissue  is  pigmented.  The  liver  and  kidneys 
are  also  enlarged  and  pigmented. 

Prophylaxis. — With  the  knowledge  of  the  role  of  the 
mosquito  in  the  transmission  of  malaria,  its  prophylaxis  be- 
comes a  matter  of  simplicity  when  certain  measures  can  be 
systematically  carried  out.  There  are  two  equally  import- 
ant factors  to  be  considered — the  human  being  and  the  mos- 
quito. The  measures  must  be  directed  toward  preventing 
each  from  infecting  the  other. 

1 .  The  Human  Beings. — In  districts  where  malarial  fever 
prevails,  the  first  part  of  the  campaign  had  perhaps  best  be 
directed  toward  finding  and  treating  all  cases  of  malarial 
fever,  so  that  the  parasites  in  their  blood  may  be  destroyed 
and  the  infection  of  mosquitoes  prevented.     This  is  done  by 
the  systematic  and  general  use  of  quinin. 

All  cases  of  malarial  fever  should  be  required  to  sleep  in 
mosquito-proof  houses  under  nets,  and  as  the  mosquitoes  are 
nocturnal  and  begin  to  fly  at  dusk,  the  patients  should  shut 
themselves  in  at  that  time.  By  thus  killing  the  parasites  in 
the  blood,  and  keeping  the  mosquitoes  from  the  patients  in 
the  meantime,  much  can  be  done.  But  where  malarial 
fever  prevails,  the  mosquitoes  are  already  largely  infected, 
hence  the  healthy  population  should  also  learn  to  respect  the 
habits  of  the  insects  and  not  expose  themselves  to  their  bites, 
should  screen  their  houses  and  their  beds,  and  should  take 
small  prophylactic  doses  of  quinin  to  prevent  the  develop- 
ment of  the  parasites  when  exposure  cannot  be  prevented. 

2.  The  Mosquitoes. — It  is  not  known  that  the  parasites 
can  pass  from  one  generation  of  mosquitoes  to  another, 
hence  the  mosquitoes  to  be  feared  are  those  that  are  already 
infected.     By  making  the  houses  mosquito-proof  most  of 
the  insects  can  be  kept  out,  while  those  that  get  in  can  be 
caught  and  killed. 

By  draining  the  swamps  and  destroying  all  the  breed- 
ing places  in  and  near  human  habitations,  the  number 


Mosquitoes  and  Malarial  Fever  541 

of  mosquitoes  can  be  greatly  diminished.  Fortunately 
this  is  particularly  true  with  reference  to  the  mosquitoes 
most  concerned — the  anopheles — which  fly  but  short  dis- 
tances. By  closing  all  the  domestic  cisterns  and  reservoirs, 
cesspools,  etc.,  so  that  no  mosquitoes  can  get  in  to  breed 
or  get  out  to  bite,  and  by  draining  the  pools  for  half  a  mile 
in  all  directions  from  human  habitations,  the  number  of 


Fig.  182. — Anopheles  maculipennis :  Adult  male  at  left,  female  at  right 

(Howard) . 


anopheles  mosquitoes  can  be  made  almost  negligible.  If  at 
the  same  time  no  mosquitoes  are  any  longer  permitted  to 
infect  themselves  by  biting  infected  human  beings,  the 
spread  of  the  disease  must  be  greatly  restricted  or  checked. 


MOSQUITOES  AND  MALARIAL  FEVER. 

In  order  that  the  student  may  be  able  to  differentiate 
with  reasonable  accuracy  such  mosquitoes  as  come  under 
his  observation,  use  must  be  made  of  tabulations,  to  cor- 
rectly use  which,  however,  the  student  should  have  some 
familiarity  with  insect  structure  and  the  general  principles 


542 


Malaria 


of  entomology.  The  mosquitoes,  or  culicidae,  must  be  rec- 
ognized first  by  their  well-known  general  form,  and  second 
by  the  presence  of  scales  upon  some  part  of  the  head,  thorax, 
abdomen,  and  wings. 


Fig.  183 — Various  mosquitoes  in  attitudes  of  repose:  a,  Culex  pipiens; 
b,  Myzorrhynchus  pseudopictus ;  c,  Anopheles  maculipennis  (Manson). 


CLASSIFICATION  (Stitt). 

There  are  four  subfamilies  of  CULICIDJS,  differentiated  according 
to  the  palpi: 

I.  Palpi  as  long  or  longer  than  the  proboscis  in  the 

male. 

i  .  Palpi  as  long  as  the  proboscis  in  the  fe- 
male ;  proboscis  straight 

2.  Palpi  as   long  or  shorter  than  the  pro- 

boscis; proboscis  curved 

3.  Palpi  shorter  than  the  proboscis 

II.  Palpi  shorter  than  the  proboscis  in  the  male 
and  female 

Of  these  the  Anophelinae  is  the  one  family  concerned  in  the  transmis- 
sion of  malarial  fever,  so  that  it  is  important  to  be  able  to  differentiate 
the  genera  included  in  the  family. 


1.  Scales  on  head  only;  hairs  on  thorax  and  abdo- 

men. 

1.  Scales     on    wings    large    and    lanceolate. 

Palpi  only  slightly  scaled  .............  Anopheles. 

2.  Wing  scales  small,  narrow,  and  lanceolate. 

Only  a  few  scales  on  palpi  ..............  Myzomyia. 

3.  Large  inflated  wing  scales  ..............  Cycloleppteron. 

2.  Scales  on  head  and  thorax.     Scales  narrow  and 

curved.     Abdomen  with  hairs,  not  scales. 
i  .  Wing  scales  small  and  lanceolate  ........  Pyretophorus. 

3.  Scales  on  head,  thorax,  and  abdomen.     Palpi 

covered  with  thick  scales. 
i.  Abdominal  scales  on  ventral  surface  only. 
Thoracic  scales  like  hairs.     Palpi  rather 
heavily  scaled  ......................  Myzorrhynchus. 


Mosquitoes  and  Malarial  Fever  543 

2.  Abdominal  scales  narrow,  curved  or  spindle 

shaped,  in  tufts  and  dorsal  patches.  .  .    Nyssorrhynchus. 

3.  Abdomen  almost  completely  covered  with 

scales  and  also  having  lateral  tufts  ....  Cellia. 

4.  Abdomen  completely  scaled  .............  Aldrichia. 

Species  of  the  genera  Anopheles,  Myzomyia,  and  Myzorrhynchus,  are 
known  to  transmit  malarial  parasites.  The  culicinae  include  Stegomyia 
and  Culex,  which  have  some  medical  interest,  as  the  former  transmits 
yellow  fever;  the  latter,  filarial  worms. 


I.  Posterior  cross-vein  nearer  the  base  of  the 

wing  than  the  mid-cross-vein. 
i  .  Proboscis  curved  in  the  female  ........  Psorophora. 

2.  Proboscis  straight  in  the  female: 

A.  Palpi  with  three  segments  in  the 

female. 

a.  Third    segment        somewhat 

longer  than  the  first  two  .  .  Culex. 

b.  The  three  segments  are  equal 

in  length  ................  Stegomyia. 

B.  Palpi  with   four  segments   in   the 

female. 

a.  Palpi  shorter  than  the  third 

of   the  proboscis.     Spotted 

wings  ...................  Theobaldia. 

b.  Palpi  longer  than  the  third  of 

the    proboscis.        Irregular 

scales  on  the  wings  .......  Mansonia. 

C.  Palpi   with   fine   segments   in   the 

female  .............  ..........  Taniorrhynchus. 

II.  Posterior  cross-  vein  in  line  with  the  mid-cross- 

vein  .................................  Joblotina. 

III.  Posterior  cross-vein  further  from  the  base  of 

the  wing  than  the  mid-cross-vein  ......  Mucidus. 

The  mosquitoes  used  for  study  and  for  classification 
should  be  mounted  dry  in  the  usual  way  well  known  to  all 
entomologists.  Fine  entomologic  pins  (oo-ooo)  should  be 
employed  for  the  purpose.  The  insects  should  be  caught  in 
a  wide-mouth  bottle  containing  some  fragments  of  cyanid  of 
potassium,  covered  with  a  layer  of  saw-dust,  over  which  a 
thin  layer  of  plaster  of  Paris  is  allowed  to  solidify.  The 
insects  die  in  a  moment  or  two,  can  be  emptied  upon  a  table, 
and  the  pin  carefully  thrust  through  the  central  part  of  the 
thorax.  As  soon  as  the  insect  is  impaled,  the  pin  should  be 
passed  through  an  opening  in  a  card  or  between  the  blades  of 
a  forceps  until  the  insect  occupies  a  position  at  the  junction 
of  the  middle  and  upper  third.  The  insect  should  not  be 
touched  with  the  fingers,  as  the  scales  will  be  brushed  off  and 
the  limbs  broken.  Mounted  insects  must  be  handled  with 


544 


Malaria 


entomologic  forceps,  touching  the  pins  only.  Every  in- 
sect thus  mounted  should  have  placed  upon  the  pin,  at  the 
junction  of  the  middle  and  lower  thirds,  a  small  bit  of  card  or 


Fig.  184. — Method  of  withdrawing  the  digestive  tube  of  the  mosquito 
for  study   (Blanchard). 

paper,  telling  where  and  when  and  under  what  circumstances 
it  was  taken. 

The  dissection  of  fresh  mosquitoes  for  determining  whether 
or  not  they  are  infected  with  malarial  organisms  must  be 
made  with  the  aid  of  needles  mounted  in  handles.  The 
position  of  the  stomach,  intestines,  and  the  salivary  glands, 


Fig.  185.- 


-Method  of  withdrawing  the  salivary  glands  of  the  mosquito 
for  study  (Blanchard). 


and  the  mode  of  pulling  the  insect  apart  to  show  them  can  be 
learned  from  the  diagram.  The  organs  thus  withdrawn  and 
separated  from  the  unnecessary  tissue  can  be  fixed  to  a  slide 
with  Meyer's  glycerin-albumin  or  other  albuminous  matter, 
and  then  stained  like  a  blood-smear,  but  should  be  cleared 


Mosquitoes  and  Malarial  Fever  545 

after  staining  and  washing,  and  mounted  in  Canada  balsam 
under  a  cover-glass. 

A  more  certain  and  more  elegant  manner  of  showing  the 
parasites  in  infected  mosquitoes  is  by  pulling  off  the  legs  and 
wings  and  then  embedding  the  insect  in  paraffin  and  cutting 
serial  longitudinal  vertical  sections. 

To  infect  mosquitoes  and  study  the  development  of  the 
malarial  parasites  in  their  bodies,  the  insects  should  be 
bred  from  the  aquatic  larva  in  the  laboratory,  to  make  sure 
that  they  do  not  already  harbor  parasites.  The  mosquitoes 
are  allowed  to  enter  a  small  cage  made  with  mosquito  netting, 
and  are  taken  to  the  bedside  of  the  malarial  patient,  against 
whose  skin  the  cage  is  placed  until  the  insects  have  bitten 
and  distended  themselves  with  blood,  when  they  are  taken 
back  to  the  laboratory  and  kept  as  many  days  as  may  be 
desired,  then  killed  and  sectioned.  In  this  way,  remember- 
ing that  the  entire  mosquito  cycle  of  development  takes 
about  a  fortnight,  any  stage  of  the  cycle  may  be  observed. 

35 


CHAPTER  XIX. 

RELAPSING  FEVER. 

SPIROCH^TA 


IN  1868  Obermeier*  first  observed  the  presence  of  actively 
motile  spiral  organisms  in  the  blood  of  a  patient  suffering 
from  relapsing  fever.  Having  made  the  observation,  he 
continued  to  study  the  organism  until  1873,  when  he  made 
his  first  publication.  From  1873  until  1890  it  was  supposed 
that  spirochaeta  rarely  played  any  pathogenic  role.  Miller 
had,  indeed,  called  attention  to  the  constant  presence  of 
Spirochaeta  dentinum  in  the  human  mouth,  but  it  had  not 
been  connected  with  any  morbid  condition.  In  1890 
Sacharofff  discovered  a  spirillary  infection  of  geese  in  the 
Caucasus,  caused  by  an  organism  much  resembling  Spiro- 
chaeta obermeieri  and  called  Spirochaeta  anserinum.  In 
1903  Marchoux  and  Salimbenif  found  a  third  disease, 
fatal  to  chickens,  caused  by  Spirochaeta  gallinarum,  and 
found  that  the  spread  of  the  disease  was  determined  by 
the  bites  of  a  tick,  Argas  miniatus.  In  1902  Theiler,§ 
in  the  Transvaal,  observed  a  spiral  organism  in  a  cattle 
plague.  This  has  been  named  after  him  by  Laveran, 
Spirochaeta  theileri.  It  was  found  to  be  disseminated  by 
the  bites  of  certain  ticks  —  Rhipicephalus  decolor  atus. 
Later,  what  was  probably  the  same  organism,  was  found  in 
the  blood  of  sheep  and  horses.  In  1905  Nicolle  and  Comte|| 
found  a  spiral  organism  infecting  certain  bats.  By  this 
time,  therefore,  it  became  evident  that  spirochaetal  infections 
were  fairly  well  disseminated  among  the  lower  animals  and 
that  the  spirochaeta  were  of  different  species  with  different 
hosts  and  intermediate  hosts. 

*"Centralbl.  f.  d.  med.  Wissenschaft,"  1873. 
t  "Ann.  de  1'Inst.  Pasteur,"  1891,  xvi,  No.  9,  p.  564. 
J  Ibid,  1903,  xvii,  p.  569. 

§  "Jour.  Comp.  Path,  and  Therap.,"  1903,  XLVII,  p.  55. 
||  "Compt.-rendu  de  la  Soc.  de  Biol.  de  Paris,"  July  22,  1905,  Lix, 
p.  200. 

546 


Relapsing  Fever  547 

In  1904  Ross  and  Milne*  and  Button  and  Toddf  studied 
a  peculiar  African  fever  which  they  were  able  to  refer  to  a 
spirochaeta  for  which  NovyJ  has  proposed  the  name  Spiro- 
chaeta  duttoni  in  memory  of  Dutton,  who  lost  his  life  while 
studying  it.  It  was  found  that  this  organism,  like  most  of 
the  others  described,  was  transmitted  by  a  tick,  Ornitho- 
doros  moubata. 

With  the  work  of  Schaudinn  and  his  associate,  Hoffmann,  § 
the  spirochaeta  came  to  be  regarded  as  protozoan  parasites 
because  of  the  presence  of  an  undulating  membrane;  the 
refusal  of  most  of  the  organisms  to  grow  upon  artificial  media, 


Fig.  1 86. — Spirochaeta  obermeieri  from  human  blood  (Kolle  and  Was- 

sermann). 

the  role  of  an  intermediate  host  (ticks,  etc.)  in  transmitting 
them,  and  the  longitudinal  mode  of  division. 

Fevers  characterized  by  relapses  and  by  the  presence  of 
spirochaeta  in  the  blood  have  been  found  in  northern  and 
northeastern  Europe  (true  relapsing  fever  with  Spirochaeta 
obermeieri),  in  various  parts  of  Africa  (African  relapsing 
fever  with  Spirochaeta  duttoni),  in  Bombay,  and  in  America. 
The  question,  therefore,  arises  whether  these  similar  diseases 
are  slight  modifications  of  the  same  thing  caused  by  the  same 

*  ''British  Med.  Jour.,"  Nov.  26,  1904,  p.  1453. 

t" Memoir  xvu,  Liverpool  School  of  Tropical  Medicine,"  "Brit. 
Med.  Jour.,"  Nov.  n,  1905,  p.  1259. 

J  "Jour.  Infectious  Diseases,"  1906,  m,  p.  295. 

§"  Deutsche  med.  Wochenschrift,"  Oct.,  1905,  xxxi,  p.  1665; 
"Arbeiten  aus  dem  kaiserlichen  Gesundheitsamte,"  1904,  xx,  pp.  387- 
439- 


548  Relapsing  Fever 

parasite,  or  whether  they  are  different  diseases  caused 
by  slightly  different  parasites.  Fulleborn,  Mayer,  and 
Martin*  consider  them  to  be  four  different  organisms: 

Spirochaeta  obermeieri,  of  the  European  relapsing  fever. 

Spirochaeta  duttoni,  of  the  African  relapsing  fever. 

Spirochaeta  novyi,  of  American  relapsing  fever. 

Spirochaeta  carteri,  of  Bombay  relapsing  fever. 

As  the  differences  between  these  organisms  are  minute,  it 
scarcely  seems  well  to  devote  space  to  the  consideration  of 


Fig.    187. — Spirochaeta    obermeieri    (Novy).     Rat    blood    No.    32  la. 

X  1500. 

each,  but  better  to  select  the  oldest  and  the  best  known— 
Spirochaeta  obermeieri — as  the  type,  describe  it,  and  point  out 
such  variations  as  are  shown  by  its  close  relations. 

General  Characteristics. — An  elongate,  flexible,  flagellated,  non- 
sporogenous,  actively  motile  spiral  organism,  pathogenic  for  man  and 
monkeys,  not  susceptible  of  cultivation  in  artificial  media,  stained  by 
ordinary  methods,  but  not  by  Gram's  method. 

Morphology. — The  Spirochaeta  obermeieri   is  extremely 

slender,  flexible,  spirally  coiled,  like  a  corkscrew,  and  pointed 

at  the  ends.    It  measures  approximately  i  ^  in  breadth  and 

10,  20,  or  even  40  ^  in  length.     The  number  of  spiral  coils 

*  "Med.  Klinik,"  No.  17,  April,  1907. 


Morphology  549 

varies  from  6  to  20;  the  diameter  of  the  coils  varies  so  greatly 
that  scarcely  any  two  are  uniform.  Wladimiroff*  doubts  the 
existence  of  a  flagellum,  but  flagella-like  appendages  are 
usually  to  be  seen  at  one  or  both  ends  of  the  organisms. 
An  undulating  membrane  attached  nearly  the  entire  length 
of  the  organism,  very  narrow,  and  inconspicuous,  forms  the 
chief  means  of  locomotion.  The  organism  is  actively  motile, 
and  darts  about  in  fresh  blood  with  a  double  movement,  con- 
sisting of  rotation  about  the  long  axis  and  serpentine  flexions. 
No  structure  can  be  made  out  by  our  present  methods  of 


Fig.  1 88. — Spirochaeta   duttoni    (Novy;.     Tick   fever,  No.  520.     Rat 
blood.      X  1500. 

staining  and  examining  the  spirochaeta.  No  spores  are 
found.  Multiplication  is  thought  to  take  place  by  longitudi- 
nal division,  though  some  believe  the  division  to  be  trans- 
verse. 

The  Spirochaeta  duttoni  is  said  by  Koch,f  in  his  interest- 
ing studies  of  "African  Relapsing  Fever,"  to  resemble  the 
Spirochaeta  obermeieri  in  all  particulars. 

The  Spirochaeta  novyi  with  which  Novy  and  Knapp  t  exper- 
imented, and  which  they  believed  to  be  identical  with 

"Kolle  and  Wassermann's  Handbuch  der  pathogene  Mikroorgan- 
ismen,"  1903,  in,  p.  82. 

f  ''Berliner  klin.  Wochenschrift,"  Feb.  12,  1906,  xxxiv,  No.  7,  p.  185. 
J  "Jour.  Infectious  Diseases,"  1906,  in,  p.  291. 


550  Relapsing  Fever 

Spirochaeta  obermeieri,  measured  0.25  to  0.3  f*  in  breadth  by 
7  to  19  ^  in  length.  The  number  of  coils  varies  from  three 
to  six.  The  shorter  forms  are  pointed,  with  a  long  flagel- 
lum  at  one  end  and  a  short  one  at  the  other. 

Staining. — The  spirochaeta  can  be  stained  with  ordinary 
anilin  dye  solutions,  by  the  Romanowsky  and  Giemsa 
methods,  and  by  the  silver  methods  (see  Treponema  palli- 
dum).  It  does  not  stain  by  Gram's  method. 

Cultivation. — The  organism  will  not  grow  upon  any 
known  culture-medium.  Following  the  suggestion  of 
Levaditi,  Novy  and  Knapp*  cultivated  Spirochaeta  ober- 
meieri in  collodion  sacs  in  the  abdominal  cavity  of  rats, 
and  succeeded  in  maintaining  it  alive  in  this  way  through 
twenty  consecutive  passages  during  sixty-eight  days.  They 
were  able  to  do  this  in  rat  serum  from  which  all  corpuscles 
had  been  removed  by  centrifugation,  so  that  it  is  proved 
that  no  intercellular  developmental  stage  of  the  organism 
takes  place.  Organisms  thus  cultivated  are  attenuated  in 
virulence. 

Norris,  Pappenheimer,  and  Flournoyf  believe  that  they 
succeeded  in  securing  multiplication  of  the  spirochaeta  by 
placing  several. drops  of  blood  containing  them  in  3  to  5  c.c.  of 
citrated  rat  or  human  blood.  A  third  generation  always 
failed. 

Mode  of  Infection. — The  means  by  which  Spirochaeta 
obermeieri  is  transmitted  from  individual  to  individual  is  not 
definitely  known.  TictinJ  seems  to  have  been  the  first  to 
believe  that  the  transmission  of  the  disease  was  accomplished 
through  the  intermediation  of  some  blood-sucking  insect. 
He  investigated  lice,  fleas,  and  bed-bugs,  in  the  latter  of 
which  he  was  able  to  find  the  organisms,  and  through  blood 
obtained  from  which  he  was  able  to  transmit  the  disease  to 
an  ape.  He  was  not  able  to  infect  apes  by  permitting  infected 
bed-bugs  to  bite  them.  Breinl  and  Kinghorn  and  Todd§ 
made  a  careful  study  of  the  subject,  but,  like  Tictin  and 
their  other  predecessors,  were  unable  to  infect  monkeys  by 
permitting  infected  bed-bugs  to  bite  them. 

This  leaves  the  transmission  of  the  micro-organism  un- 
accounted for.  When  we  come  to  consider  Spirochaeta 

*  "Jour.  Amer.  Med.  Assoc.,"  Dec.  29,  1906,  XLVII,  p.  2152. 
t  "Journal  of  Infectious  Diseases,"  1906,  in,  266. 
J  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  i  Abt.,  xv,  1894,  P-  840. 
§  Ibid.,  Oct.,  1906,  xui,  Heft  6,  p.  537. 


Mode  of  Infection  551 

duttoni,  however,  we  find  our  knowledge  much  further 
advanced.  On  Nov.  26,  1904,  Button  and  Todd  announced 
that  they  had  discovered  a  spirillum  to  be  the  specific 
agent  in  the  causation  of  tick  fever  in  the  Congo,  and  on 
the  same  date  Ross  and  Milne*  published  the  same  fact. 
Button  and  Todd  subsequently  withdrew  their  claim  to 
priority  of  the  discovery.  On  Feb.  4,  1905,  Ross  published 
in  the  "British  Medical  Journal"  the  following  cablegram 
from  Button  and  Todd,  then  working  on  the  Congo:  "Spirilla 
cause  human  'tick  fever;  naturally  infected  ornithodoros 
infect  monkey."  It  was  not  until  Nov.  n,  1905,  that  the 
paper  upon  the  subject  was  read  and  published  in  the  same 


Fig.  189. — Ornithodorus  moubata.  Tick  that  transmits  African 
relapsing  fever:  a,  Viewed  from  above;  b,  viewed  from  below  (Murray 
from  Doflein). 

journal  by  Button  and  Todd,  and  the  etiology  of  the  disease 
made  clear.  These  observers  found  that  the  horse- tick, 
Ornithodoros  moubata  (Murray)  is  the  intermediate  host  of 
the  spirilla  or  spirochaeta  causing  the  disease,  and  that  when 
these  ticks  were  permitted  to  bite  infected  human  beings, 
and  then  subsequently  transferred  to  monkeys,  the  latter 
sickened  with  the  typical  infection. 

The  matter  received  confirmation  and  addition  through 
the  studies  of  Koch,f  who  studied  the  ticks,  observed  the 
distribution  of  the  micro-organisms  in  their  bodies,  and 
found  that  they  collected  in  large  numbers  in  the  ovaries, 

*  "British  Medical  Journal,"  Nov.  26,  1904. 
t  "Berliner  klin.  Wochenschrift,"  Feb.  12,  1906. 


552  Relapsing  Fever 

so  that  the  eggs  were  commonly  infected  and  the  embryo 
hexapod  ticks  hatched  from  them  were  infective.  Thus, 
in  regard  to  Spirochaeta  duttoni  we  are  able  to  say  quite 
definitely  that  the  tick  is  the  usual  if  not  the  only  means  of 
dissemination. 

Pathogenesis. — The  spirochaeta  of  relapsing  fever  are 
pathogenic  for  man  and  monkeys,  some  of  them  for  smaller 
animals.  Novy  and  Knapp*  found  their  organism  and  Spiro- 
chseta  duttoni  to  be  infectious  for  mice  and  rats,  and  attribute 
the  failure  of  others  to  discover  this  to  their  failure  to  exam- 
ine the  blood  during  the  first  and  second  days.  Fulleborn 
and  Meyer  and  Martin  f  were  able  successfully  to  transmit 
the  spirochaeta  of  Russian  relapsing  fever  to  mice  after  first 
passing  it  through  apes.  Rabbits  and  guinea-pigs  seem 
to  be  refractory;  white  mice  susceptible.  Man,  monkeys, 
and  mice  suffer  from  infection  characterized  by  relapses, 
and  in  them  the  disease  may  be  fatal.  Rats  never  die  and 
rarely  have  relapses. 

The  micro-organisms  are  free  parasites  of  the  blood  in 
which  they  swim  with  a  varying  rapidity,  according  to  the 
stage  of  the  disease.  They  are  present  during  the  febrile 
paroxysms  only,  disappearing  completely  as  soon  as  the 
crisis  is  reached. 

The  course  of  relapsing  fever  in  man  is  peculiar  and  char- 
acteristic. After  a  short  incubation  period  the  invasion 
comes  on  with  chill,  fever,  headache,  pain  in  the  back, 
nausea  and  vomiting,  and  sometimes  convulsions.  The  tem- 
perature rises  rapidly  and  there  are  frequent  sweats.  The 
pulse  is  rapid.  By  the  second  day  the  temperature  may  be 
104°  to  105°  F.  and  the  pulse  1 10  to  130.  There  is  enlargement 
of  the  spleen.  Icteroid  discoloration  of  the  conjunctiva  may 
be  observed.  The  fever  persists  with  severity  and  the 
patient  appears  very  ill  for  five  or  six  days,  when  a  crisis  occurs, 
and  the  temperature  returns  to  normal;  there  is  profuse 
sweating  and  sometimes  marked  diarrhea,  and  the  patient  at 
once  begins  to  improve.  So  rapid  is  the  convalescence  that 
in  a  few  days  he  may  be  up  and  may  desire  to  go  out.  The 
disease  is,  however,  not  at  an  end,  for  on  or  about  the  four- 
teenth day  the  relapse  characteristic  of  the  affection  makes 
its  appearance  as  an  exact  repetition  of  what  has  gone  be- 
fore. This  is  followed  by  another  apyretic  interval,  and 
then  by  another  relapse,  and  so  on.  The  patient  usually  re- 
*  Loc.  cit.  t  Loc.  cit. 


Immunity  553 

covers,  the  mortality  being  about  4  per  cent.  The  fatal 
cases  are  usually  old  or  already  infirm  patients.  The  Indian, 
African,  and  American  varieties  present  variations  of  no  great 
importance.  The  European  fever  usually  ends  after  the  sec- 
ond or  third  relapse,  the  African  not  until  after  a  greater 
number. 

The  spirochaeta  are  present  in  the  blood  in  great  numbers 
during  the  febrile  stages,  but  entirely  disappear  during  the 
intervals: 

Lesions. — There  are  no  lesions  characteristic  of  relapsing 
fever. 

Bacteriologic  Diagnosis. — This  should  be  quite  easily 
made  by  an  examination  of  either  the  fresh  or  stained 
blood,  provided  the  blood  be  secured  during  a  febrile  parox- 
ysm. The  readiness  with  which  the  organisms  take  the  stain 
leaves  little  to  be  desired. 

Novy  and  Knapp  have  found  that  the  serum  of  recovered 
cases  can  be  used  to  assist  in  making  diagnosis  because  of  its 
agglutinating,  germicidal,  and  immunizing  powers. 

Immunity. — The  phenomena  of  immunity  are  vivid  and 
important.  At  the  moment  of  decline  of  the  fever  a  power- 
ful bacteriolytic  substance  appears  in  the  blood  and  dis- 
solves the  organisms.  At  the  same  time  an  immunizing 
substance  appears.  The  two  do  not  appear  to  be  the  same. 

The  immunizing  body  affords  future  protection  to  the 
individual  for  an  indefinite  length  of  time.  It  can  be 
increased  by  rapidly  injecting  the  animal  with  blood  con- 
taining spirochaeta.  Serum  containing  the  immunizing  body 
imparts  passive  immunity  to  other  animals  into  which  it  is 
injected,  and,  according  to  Novy  and  Knapp,  establishes  a 
solid  basis  for  the  prevention  and  cure  of  relapsing  fever  in 
man. 


CHAPTER  XX. 

SLEEPING  SICKNESS. 

TRYPANOSOMA  GAMBIENSE  (BUTTON,  1902). 

SLEEPING  sickness,  African  lethargy,  Maladie  du  sommeil, 
Schlafkrankheit,  or  human  trypanosomiasis  is  a  specific,  in- 
fectious, endemic  disease  of  equatorial  Africa  characterized 
by  fever,  lassitude,  weakness,  wasting,  somnolence,  coma, 
and  death.  The  first  mention  of  the  disease  seems  to  have 
been  made  by  Winterbottom.* 

Sir  Patrick  Mansonf  says  that  "For  upward  of  a  century 
students  of  tropical  pathology  have  puzzled  over  a  peculiar 
striking  African  disease,  somewhat  inaccurately  described  by 
its  popular  name,  the  sleeping  sickness.  Its  weirdness  and 
dreadful  fatality  have  gained  for  it  a  place  not  in  medical 
literature  only,  but  also  in  general  literature.  The  mystery 
of  its  origin,  its  slow  but  sure  advance,  the  prolonged  life  in 
death  that  so  often  characterizes  its  terminal  phases,  and  its 
inevitable  issue,  have  appealed  to  the  imagination  of  the 
novelist,  who  more  than  once  has  brought  it  on  his  mimic 
stage,  draping  it,  perhaps,  as  the  fitting  nemesis  of  evil-doing. 
The  leading  features  of  the  strange  sickness  are  such  as  might 
be  produced  by  a  chronic  meningo-encephalitis.  Slow  irreg- 
ular febrile  disturbance,  headache,  lassitude,  deepening  into 
profound  physical  and  mental  lethargy,  muscular  tremor, 
spasm,  paresis,  sopor,  ultimately  wasting,  bed-sores,  and 
death  by  epileptiform  seizure,  or  by  exhaustion,  or  by  some 
inter  current  infection. 

"  In  every  case  the  lymphatic  glands,  especially  the  cer- 
vical, are  enlarged,  though  it  be  but  slightly.  In  many  cases 
pruritus  is  marked.  In  all,  lethargy  is  the  dominating  feature. 

"In  some  respects  this  disease,  which  runs  its  course  in 
from  three  months  to  three  years  from  the  oncoming  of  the 

*  "An  Account  of  Native  Africans  in  the  Neighborhood  of  Sierre 
Leone,"  1803. 

t  "The  Lane  Lectures  for  1905,"  Chicago,  1905. 

554 


Specific  Organism  555 

decided  symptoms,  resembles  the  general  paralysis  of  the 
insane.  It  differs  from  this,  however,  in  the  absence,  as  a 
rule,  of  the  peculiar  psychic  phenomenon  of  that  disease. 
There  are  exceptions,  but  generally,  though  the  mental  fac- 
ulties in  sleeping  sickness  are  dull  and  slow  acting,  the 
patient  has  no  mania,  no  delusions,  no  optimism.  So  far 
is  the  last  from  being  the  case,  that  he  is  painfully  aware 
of  his  condition  and  of  the  miserable  fate  that  is  in  store  for 
him;  and  he  looks  as  if  he  knew  it." 

Specific  Organism. — The  discovery  of  the  specific  organ- 
isms was  foreshadowed  by  Nepveu,*  who  recorded  the  exist- 
ence of  trypanosomes  in  the  blood  of  several  patients  coming 
from  Algeria,  by  Barron,  f  and  by  Brault.f  In  1901  Forde 
received  under  his  care  at  the  hospital  in  Bathurst  (Gambia), 


i 


>K 


t 


<*: 


Fig.  190. — Trypanosoma  gambiense  (Todd). 

a  European,  the  captain  of  a  steamer  on  the  River  Gambia,  who 
had  navigated  the  river  for  six  years,  and  who  had  suffered  sev- 
eral attacks  of  fever  that  were  looked  upon  as  malarial.  The 
examination  of  his  blood  revealed  the  presence  not  of  malarial 
parasites,  but  of  small  worm-like  bodies,  concerning  the  nature 
of  which  Forde  was  undecided.  §  Later,  Button,  in  conjunc- 
tion with  Forde,  examined  this  patient,  whose  condition  had 
become  more  serious,  and  recognized  that  these  worm-like 
bodies  seen  by  Forde  were  trypanosomes.  Of  these  parasites 

*  "Memoirs,  Soc.  de  Biol.  de  Paris,"  1891,  p.  49. 
t  "Transactions  of  the  Liverpool  Medical  Institute,"  Dec.  6,  1894. 
t  "Janus,"  July  to  August,  1898,  p.  41. 

§  "Trypanosomes    and    Trypanosomiasis,"    Laveran    and    Mesnil, 
1907. 


556 


Sleeping  Sickness 


he  has  written  an  excellent  description,  calling  them  Try- 
panosoma  gambiense.*     The  patient  thus  studied  by  Forde 


Fig.  191. — Various  species  of  trypanosoma:  i,  Trypanosoma  lewisi 
of  the  rat;  2,  Trypanosoma  lewisi,  multiplication  rosette;  3,  Trypano- 
soma lewisi,  small  form  resulting  from  the  disintegration  of  a  rosette; 
4,  Trypanosoma  brucei  of  nagana;  5,  Trypanosoma  equinum  of  caderas; 
6,  Trypanosoma  gambiense  of  sleeping  sickness;  7,  Trypanosoma  gam- 
biense, undergoing  division;  8,  Trypanosoma  theileri,  a  harmless  trypano- 
some  of  cattle;  9,  Trypanosoma  transvaliense,  a  variation  of  T.  theileri; 
10,  Trypanosoma  amum,  a  bird  trypanosome;  1 1,  Trypanosoma  damonice 
of  a  tortoise;  12,  Trypanosoma  solece  of  the  flat  fish;  13,  Trypanosoma 
granulosum  of  the  eel;  14,  Trypanosoma  rajas,  of  the  skate;  15,  Trypano- 
soma rotatorium  of  frogs;  16,  Cryptobia  borreli  of  the  red-eye  (a  fish). 
(From  Laveran  and  Mesnil.) 

*  See  Forde,  "Jour.  Trop.  Med.,"  Sept.  i,  1902;  Button,  Ibid.,  Dec. 
i,  1902;  Button,  "Thompson  Yates'  Laboratory  Reports,"  1902,  v,  4, 
part  n,  p.  455. 


Specific  Organism  557 

and  Dutton  died  in  England  January  i,  1903.  In  1903 
Button  and  Todd*  examined  1000  persons  in  Gambia  and 
found  similar  trypanosomes  in  the  bloods  of  6  natives  and 
i  quadroon.  In  the  same  year  Mansonf  discovered  2  cases 
of  trypanosomiasis  in  Europeans  that  had  become  infected 
upon  the  Congo.  Brumptj  also  observed  T.  gambiense  at 
Bounba  at  the  junction  of  the  Ruby  and  the  Congo,  and 
Baker  observed  3  cases  at  Entebbe  in  Uganda.  § 

During  all  this  time  no  connection  was  suspected  between 
these  micro-organisms  and  African  lethargy,  and  much  inter- 
est was  being  taken  in  a  coccus — the  hypnococcus — that 
was  being  studied  by  Castellani  in  Uganda.  As  Castellani 
was  prosecuting  the  investigation  of  this  organism,  he  chanced 
to  examine  the  cerebrospinal  fluid  of  several  negroes  in 
Uganda  who  were  suffering  from  sleeping-sickness,  and  in  it 
found  trypanosomes.  Even  then,  though  Castellani  ||  real- 
ized that  these  organisms  were  connected  with  sleeping-sick- 
ness, he  did  not  identify  them  in  his  mind  with  the  Trypano- 
soma  gambiense  discovered  in  the  blood  by  Forde  and  Dut- 
ton, and  described  the  newly  discovered  organism  as  Try- 
panosoma  ugandense.  Kruse,**  thinking  to  honor  the  dis- 
coverer, called  it  Trypanosoma  castellani.  Bruce  and 
Nabarroft  found  the  new  trypanosome  in  each  of  38  cases  of 
sleeping  sickness  in  the  cerebrospinal  fluid,  and  12  out  of  13 
times  in  the  blood.  These  observers  also  found  that  23  out 
of  28  natives  from  parts  of  Uganda  where  sleeping  sickness 
is  endemic  had  trypanosomes  in  their  blood,  while  in  117 
natives  from  uninfected  areas  the  blood  examination  was  neg- 
ative in  every  case.  They  also  declared  that,  contrary  to 
what  had  been  stated,  there  were  no  appreciable  morphologic 
differences  between  Trypanosoma  gambiense  and  Trypano- 
soma ugandense.  Dutton,  Todd,  and  Christy  tJ  arrived  at 
the  same  conclusion.  The  matter  was  finally  settled  by 

*  "First  Report  of  the  Trypanosomiasis  Expedition  to  Senegambia," 
1902,  Liverpool,  1903. 

t"Jour.  Trop.  Med.,"  Nov.  i,  1902,  and  March  16,  1903;  "Brit. 
Med.  Jour.,"  May  30,  1903. 

I  "Acad.  de  Med.,"  March  17,  1903. 

§  "Brit.  Med.  Jour.,"  May  30,  1903. 

||  Ibid.,  May  23,  1903;  June  20,  1903. 

**"Gesell.  f.  natur.  Heilkunde,"  1903. 

ft  "Brit.  Med.  Jour.,"  Nov.  21,  1903. 

Jt  Ibid.,  Jan.  23,  1904,  also  " Thompson- Yates  and  Johnson  Lab. 
Reports,"  v,  6,  part  i,  1905,  pp.  1-45. 


558  Sleeping  Sickness 

Thomas  and  Linton*  and  Laveran,|  who,  by  means  of 
animal  experiments,  determined  not  only  the  complete 
identity  of  the  organisms,  but  their  uniform  virulence. 

Morphology. — Trypanosoma  gambiense  is  a  long,  slender, 
spindle-shaped,  flagellate  micro-organism  that  measures  17 
to  28  ^  in  length  and  1.4  to  2  ^  in  breadth.  From  the  ante- 
rior end  (that  which  moves  forward  as  the  organism  swims) 
a  whip-like  flagellum  projects  about  half  the  length  of  the 
organism.  The  terminal  third  of  the  flagellum  is  free  in 
most  cases.  The  proximal  two-thirds  are  connected  with  a 
band  of  the  body  substance,  which  is  continued  like  a  ruffle 
along  one  side  of  the  organism  to  within  a  short  distance  of 
its  blunt  posterior  end,  where  the  flagellum  abruptly  ends 
at  the  blepharoplast.  This  thin  ruffle  is  known  as  the  un- 
dulating membrane.  By  means  of  the  flagellum  and  the 
undulating  membrane  the  organism  swims  rapidly  with  a 
wriggling  and  rotary  movement  that  give  it  the  name  Try- 
panosome,  which  means  "  boring  body." 

The  protoplasm  is  granular  and  often  contains  chromatin 
dots  that  are  remarkable  for  their  size  and  number.  There 
is  a  distinct  nucleus  of  ovoid  form.  There  is  also  a  cen- 
trosome  or  blepharoplast,  which  appears  as  a  distinct  deeply 
staining  clot  near  the  posterior  blunt  end  and  from  which  the 
flagellum  appears  to  arise.  Near  this  a  vacuole  is  sometimes 
situated. 

Staining. — The  organisms  are  best  observed  when  stained 
with  one  of  the  polychrome  methylene-blue  combinations — 
Irishman's,  Wright's,  Jenner's,  Romanowsky's,  Marino's. 
To  stain  them  a  spread  of  the  blood  or  cerebrospinal  fluid  is 
made  and  treated  precisely  as  though  staining  the  blood  for 
the  differential  leukocyte  count  or  for  the  malarial  parasite. 

Cultivation. — The  cultivation  of  Trypanosoma  gam- 
biense has  not  yet  been  achieved.  This  seems  singular,  as 
Trypanosoma  lewisi  of  the  rat  and  Trypanosoma  brucei  of 
"nagana"  or  "tsetse-fly"  disease  of  Africa  have  been  culti- 
vated by  Novy  and  McNealf  in  mixtures  composed  of 
ordinary  culture  agar-agar  and  defibrinated  rabbit-blood, 
combined  as  necessary,  1:1,  2:1,  1:2,  or  2  13,  etc.  The 

*  "Lancet,"  May  14,  1904,  pp.  1337-1340. 

t  "Compt.-rendu  de  1'Acad.  des  Sciences,"  v,  142,  1906,  p.  1065. 

J  "Contributions  to  Medical  Research  dedicated  to  Victor  Clarence 
Vaughan,"  Ann  Arbor,  Michigan,  1903,  p.  549;  "Journal  of  Infectious 
Diseases,"  1904,  i,  p.  i. 


Transmission  559 

actual  culture  was  made  chiefly  in  the  water  of  condensation 
collected  at  the  bottom  of  obliquely  congealed  media. 

Laveran  and  Mesnil  found  that  when  blood  containing 
Trypanosoma  gambiense  was  mixed  with  salt  solution  or 
horse-serum,  the  trypanosomes  remain  alive  for  five  or  six 
days  at  the  temperature  of  the  laboratory.  They  live  much 
longer  in  tubes  of  rabbit's  blood  and  agar,  sometimes  as  long 
as  nineteen  days,  and  during  this  time  many  dividing  forms 
but  no  rosettes  were  observed.  But  subcultures  failed,  and 
eventually  the  original  culture  died  out. 

Reproduction. — Multiplication  takes  place  by  binary 
division,  the  line  of  cleavage  being  longitudinal.  The  cen- 
trosome  and  nucleus  divide,  then  the  flagellum  divides  longi- 
tudinally, and  finally  the  protoplasm  divides. 

In  addition  to  this  simple  longitudinal  fusion,  the  trypan- 
osomes seem  to  possess  a  sexual  mode  of  reproduction.  When 
the  well-stained  organisms  are  carefully  studied,  it  is  possible 
to  divide  them  into  three  groups — those  that  are  peculiarly 
slender,  those  that  are  peculiarly  broad,  and  those  of  ordinary 
breadth.  The  fact  that  conjugation  takes  place  between  the 
first  two  has  led  to  the  opinion  that  they  represent  the  male 
and  female  gametocytes  respectively,  while  the  others  are 
asexual.  All  forms  multiply  by  fission,  and  conjugation 
between  the  gametes  is  observed  to  take  place  only  in  the 
body  of  the  invertebrate  host.  It  has  not  yet  been  accurately 
followed  in  the  case  of  Trypanosoma  gambiense,  but  there  is 
no  reason  to  think  that  the  organism  differs  in  its  method  of 
reproduction  from  Trypanosoma  lewisi.  Prowazek  found 
that  when  rat  blood  containing  the  latter  organism  was 
taken  into  the  stomach  of  the  rat  louse,  Hematopinus  spinu- 
losus,  the  male  trypanosome  enters  the  female  near  the 
micronucleus  and  the  various  parts  of  the  two  individuals 
become  fused.  A  non-flagellate  ookinete  results,  and,  after 
passing  through  a  spindle-shaped  gregarine-like  stage,  can 
develop  into  an  immature  trypanosome-like  form  in  the  cells 
of  the  intestinal  epithelium,  after  which  the  parasite  is 
thought  to  enter  the  general  body  cavity,  and,  migrating  to 
the  pharynx,  enter  the  proboscis,  through  which  it  is  trans- 
mitted to  a  fresh  host. 

Transmission. — It  is  well  known  that  the  disease  does 
not  spread  from  person  to  person.  In  the  days  when  African 
negroes  were  imported  into  America  as  slaves  the  disease 
often  reached  our  shores,  and  though  freshly  arrived  negroes 


560  Sleeping  Sickness 

and  those  in  the  country  less  than  a  year  frequently  died  of  it, 
there  was  no  spread  of  the  affection  to  those  that  were  ac- 
climated. The  Europeans  that  carried  the  disease  from 
Africa  to  England  and  were  the  first  in  whose  bloods  the 
trypanosomes  were  found,  did  not  spread  it  among  their  fel- 
low countrymen.  A  case  from  the  Congo  that  died  in  a 
hospital  in  Philadelphia  and  came  to  autopsy  at  my  hands, 
did  not  spread  the  disease  in  this  city. 

Yet  the  disease  is  infectious,  and  the  transfer  of  a  small 
quantity  of  the  parasite-containing  blood  to  appropriate 
experiment  animals  perfectly  reproduces  it. 

The  present  knowledge  of  the  mode  of  transmission  came 
about  through  the  knowledge  of  other  trypanosome  infections 
that  had  already  been  carefully  studied  and  understood. 
In  speaking  of  nagana  or  tsetse-fly  disease  Livingstone,  as 
early  as  1857,  recognized  that  the  flies  had  to  do  with  it. 
For  years,  however,  the  supposition  was  that  the  fly  was 
poisonous  and  that  its  venom  was  responsible  for  the  disease. 
In  1875  Megnin  stated  that  the  tsetse -fly  carries  a  virus,  and 
does  not  inoculate  a  poison  of  its  own.  In  1879  Drysdale  sug- 
gested that  the  fly  might  be  an  intermediate  host  of  some 
blood  parasite,  or  the  means  of  conveying  some  infectious 
poison.  In  1884  Railliet  and  Nocard,  who  suspected  the 
same  thing,  proved  that  inoculations  with  the  proboscis  of 
the  tsetse-flies  were  harmless.  The  exact  connection  be- 
tween the  flies  and  the  disease  was  worked  out  by  Bruce,* 
who  found,  first,  that  flies  fed  on  infected  animals,  kept  in 
captivity  for  several  days,  and  afterward  placed  upon  two 
dogs,  did  not  infect;  second,  that  flies  fed  on  a  sick  dog, 
and  immediately  afterward  on  a  healthy  dog,  conveyed  the 
disease  to  the  latter.  The  flies  were  infectious  for  twelve, 
twenty-four,  and  even  forty-eight  hours  after  having  fed 
on  the  infected  animal.  It  was,  therefore,  shown  that  the 
flies  could  and  did  infect,  not  through  something  of  which  they 
were  constantly  possessed,  but  through  something  taken  from 
the  one  animal  and  put  into  the  other ;  this,  of  course,  proved 
to  be  the  trypanosome.  Further,  it  was  shown  that  where 
there  were  no  tsetse -flies,  there  never  was  nagana. 

So  soon  as  African  lethargy  was  shown  to  be  a  form  of 
trypanosomiasis,  the  question  arose,  Was  it  spread  by  tsetse- 

*  "Preliminary  Report  on  the  Tsetse-fly  Disease  or  Nagana  in 
Zululand,  Ubombo,  Zululand,"  Dec.,  1895;  "Further  Report,"  etc., 
Ubombo,  May  29,  1896;  London,  1897. 


Transmission 


561 


flies  ?  Sambon*  and  Brumpt  f  both  suggested  it,  but  it  was 
soon  discovered  that  the  geographic  distribution  of  the  tsetse- 
fly,  Glossina  morsitans,  that  distributes  nagana,  does  not  co- 
incide with  the  geographic  distribution  of  sleeping  sickness. 
There  are,  however,  different  kinds  of  tsetse-flies,  and  Bruce 
and  NabarroJ  first  showed  that  it  was  not  Glossina  morsi- 
tans, but  a  different  tsetse-fly,  Glossina  palpalis,  that  is  the 
most  important,  if  not  the  only  source  of  the  spread  of  human 
trypanosomiasis.  They  submitted  a  black-faced  monkey 
(Cercopithicus)  to  the  bites  of  numerous  tsetse-flies  caught 
in  Entebbe,  Uganda,  and  found  trypanosomes  in  its  blood. 


Fig.  192. — Glossina  pal- 
palis. A  perfect  insect  just 
escaped  from  the  pupa.  B, 
pupa;  a,  b,  valves;  c,  body 
of  the  pupa  (Brumpt) . 


Fig.    193. — Glossina  palpalis  before 
and  after  feeding  (Brumpt). 


Bruce,  Nabarro,  and  Greig§  allowed  Glossina  palpalis  to  suck 
the  blood  of  negroes  affected  with  sleeping  sickness  and 
afterward  to  bite  five  monkeys  (Cercopithicus).  At  the  end 
of  about  two  months  trypanosomes  appeared  in  the  blood 
of  these  monkeys.  They  also  made  maps  showing  the  geo- 
graphic distribution  of  African  lethargy  and  of  Glossina 
palpalis,  which  were  found  perfectly  to  correspond. 

It  is,  of  course,  not  impossible  that  other  flies,  especially 
other  species  of  tsetse-flies,  may  act  as  distributing  hosts  of 

c  "Jour.  Trop.  Med.,"  July  i,  1903. 
t  "C.  R.  Soc.  de  Biol.,"  Jan.  27,  1903. 

t  ''Reports  of  the  Sleeping  Sickness  Commission  of  the  Royal  Society," 
1903,  i,  ii,  ii. 

§  Ibid.,  1903,  No.  4,  vin,  3. 
36 


562  Sleeping  Sickness 

the  trypanosomes,  but  there  is  no  doubt  about  the  chief 
agent  being  Glossina  palpalis.  With  increased  entomologic 
and  geographic  information  it  has  been  found  that  there 
are  certain  districts  where  these  flies  abound  though  the  dis- 
ease is  unknown,  but  that  only  shows  that  in  those  districts 
the  flies  are  not  infected.  Tsetse-flies  are  not,  as  was  for- 
merly supposed,  peculiar  to  Africa,  but  have  been  found  in 
Arabia,  where  African  lethargy  could  no  doubt  spread  should 
the  flies  become  infected  through  imported  cases  of  the  dis- 
ease. The  inability  of  the  disease  to  spread  in  England  and 
America  depends  upon  the  absence  of  tsetse-flies  from  those 
countries. 

According  to  recent  experiments  of  Bruce,  Harrison,  Hamer- 
ton,  Batement,  and  Mackie,  and  especially  of  Kleine,  only  a 
small  proportion  of  the  tsetse-flies  are  capable  of  spreading 
the  infection,  because  the  transmission  is  not  merely  the 
mechanical  transplantation  of  the  trypanosomes,  but  the 
implantation  of  the  parasites  after  they  have  completed  a 
certain  developmental  cycle  in  the  body  of  the  fly.  Thus, 
the  insect  does  not  become  infective  until  about  eighteen 
hours  after  infecting  itself,  and  remains  infective  seventy- 
five  days  (Brumpt).  Of  course,  to  be  infected,  one  must  be 
bitten  by  a  fly  itself  infected  and  in  the  infective  stage. 

It  is  possible  for  the  disease  tb  be  transmitted  from  human 
being  to  human  being  through  such  personal  contacts  as  may 
afford  opportunity  for  interchange  of  blood.  Thus,  Koch 
observed  that  in  certain  parts  of  Africa  where  there  were  no 
tsetse-flies  the  wives  of  men  that  had  become  infected  in 
tsetse-fly  countries  sometimes  developed  the  disease,  prob- 
ably through  sexual  intercourse,  a  probable  explanation  when 
one  remembers  that  it  is  solely  or  chiefly  by  such  means  that 
a  trypanosome  disease  of  horses — Dourine  or  Maladie  du 
coit,  caused  by  Trypanosoma  equiperdum — is  transmitted. 

Transmission  to  Lower  Animals. — Try panosoma  gambiense 
is  infectious  for  monkeys  as  well  as  for  human  beings.  In  the 
monkeys  a  disease  indistinguishable  from  the  sleeping  sickness 
is  brought  about.  It  is  also  infective  for  dogs,  cats,  guinea- 
pigs,  rabbits,  rats,  mice,  marmots,  hedgehogs,  goats,  sheep, 
cattle,  horses,  and  asses.  The  lower  animals  are  not,  how- 
ever, so  far  as  is  known,  subject  to  natural  infection. 

Pathogenesis. — The  first  effect  of  human  trypanosomiasis 
seems  to  be  fever  of  an  irregular  and  atypical  type,  occurring 
in  irregular  paroxysms.  It  was  in  such  cases  that  Forde  and 


Prophylaxis  563 

Dutton  first  found  the  parasites  in  the  blood.  As  the  para- 
sites increase  in  number  the  somnolence  begins  to  show  itself. 
The  lymph-nodes  enlarge  at  this  time  and  become  easily 
palpable,  and  their  enlargement  is  looked  upon  as  one  of  the 
most  ready  means  for  confirming  the  diagnosis  in  suspicious 
cases. 

In  the  bodies  of  those  that  die  there  are  few  changes  vis- 
ible to  the  naked  eye,  but  the  microscope  re  veals  that  through- 
out the  body  and  especially  in  the  nervous  system  there  are 
perivascular  collections  of  lymphocytes  and  many  trypano- 
somes. 

Prophylaxis. — This  must  be  partly  based  upon  measures 
taken  to  prevent  the  infection  of  men  by  the  flies,  and  partly 
upon  those  preventing  the  infection  of  the  flies  by  the  men. 
Its  success  must  depend  upon  the  probability  that  there  is 
no  other  host — wild  animals — in  which  the  parasites  are  kept 
alive  where  there  are  no  men. 

The  tsetse-fly — Glossina  palpalis — is  a  fairly  large  brown- 
ish insect,  easily  recognized  when  once  pointed  out.  It  makes 
a  loud  humming  sound  and  thus  attracts  attention  to  itself. 
In  the  larval  and  pupal  stages  it  lives  in  the  soft  mud  along 
the  banks  of  the  streams  and  rivers,  and  its  natural  prey  is 
thought  to  be  the  crocodile,  though  it  readily  turns  to  other 
animals  and  to  human  beings.  In  the  adult  state  it  still 
remains  more  or  less  confined  to  the  streams,  though  it  has 
been  found  to  fly  as  far  as  a  mile. 

To  prevent  the  infection  of  men  by  the  flies  is  extremely 
difficult  where  naked  or  half -naked  savages  are  to  be  dealt 
with.  For  Europeans,  the  customary  dress,  the  avoidance  of 
exposure  in  bathing,  the  use  of  mosquito  guards,  etc.,  are  to 
be  recommended,  as  well  as  the  erection  of  habitations  and  the 
building  of  roads,  etc.,  as  far  as  possible  from  the  fly  districts. 
The  destruction  of  the  grass  and  reeds  along  the  river  banks, 
the  use  of  drainage,  and  the  introduction  of  chickens,  to  pick 
up  the  larvae  and  pupae,  have  been  recommended. 

To  prevent  infection  of  the  flies  is  impossible  where,  as  in 
some  sections  of  Africa,  50  per  cent,  of  the  population  of 
some  of  the  villages  already  harbor  the  parasites.  A  means 
to  this  end  is,  however,  being  tried,  and  the  entire  population 
of  a  district  has  been  removed  from  the  fly  country  and  the 
land  abandoned,  in  the  hope  that  when,  after  the  lapse  of 
some  time,  only  healthy  persons  are  permitted  to  return,  the 
absence  of  infected  flies  in  the  country,  and  the  absence  of 


$64  Sleeping  Sickness 

infective  organisms  from  the  blood  of  the  healthy  persons 
newly  entering  it,  may  remove  most  of  the  danger. 

The  importance  of  undertaking  radical  measures  for  the 
suppression  of  the  disease  may  be  imagined  when  it  is  under- 
stood that  in  the  last  few  years  no  less  than  a  half -million  of 
the  natives  of  the  infected  districts  have  died  of  sleeping 
sickness. 

AMERICAN  TRYPANOSOMIASIS. 
TRYPANOSOMA  CRUZI  (CHAGAS). 

Human  trypanosomiasis  in  America  is  extremely  rare  and 
does  not  assume  the  form  of  sleeping  sickness.  The  occur- 
rences, thus  far  reported,  have  been  in  Brazil,  where  a  disease 


Fig.  194. — Conorhinus  megistus  (female),  the  insect  host  and  distributing 
agent  of  Trypanosoma  cruzi  (Chagas). 

of  childhood,  characterized  by  fever,  anemia,  edema,  enlarge- 
ment of  the  lymphatic  nodes,  liver,  and  spleen,  and  ending  in 
convulsions  and  death,  goes  by  the  name  of  "Apilacao." 
In  the  peripheral  blood  of  i  case  of  this  affection  Chagas* 
found  a  trypanosome  which  he  describes  as  Trypanosoma 

*  "Archives  fur  Schiffs  u.  Trop.  Hyg.,"  1909,  H.  4;  "Abstract  Cen- 
tralblatt.  f.  Bakt.  etc.  Ref.,"  1909,  xuv,  639;  also  "Ref.  Bull.  del'Inst. 
Pasteur,"  1910,  vin,  373. 


American  Trypanosomiasis  565 

cruzi.  It  proved  upon  experiment  to  be  infective  for  Brazil- 
ian monkeys,  dogs,  guinea-pigs,  rabbits,  and  other  small 
animals. 

It  has  been  successfully  cultivated  upon  the  special  blood 
agar-agar  of  Novy  and  McNeal. 

The  developmental  cycle  of  this  trypanosome  as  worked 
out  by  Chagas  is  peculiar,  and  not  only  takes  place  by  the 
usual  asexual  fission,  but  also  is  apparently  in  some  way 
associated  with  an  intracorpuscular  infection,  in  which  fig- 
ures resembling  the  merozoits  of  the  malarial  plasmodium 
are  formed.  These  subsequently  grow  into  trypanosomes. 
In  the  lungs,  also,  special  forms  are  said  to  develop,  con- 
taining light  bodies  resembling  the  merozoits  seen  in  the 
peripheral  circulation,  and  probably  corresponding  to  the 
bodies  seen  by  Schaudinn  in  his  studies  of  Trypanosoma 
noctua,  in  the  stomach  of  the  mosquito. 

Transmission. — The  transmitting  agent  was  found  by 
Chagas  to  be  a  rather  large  hemipterus  insect,  Conorhinus 
megistus.  A  special  cycle  of  development  of  the  parasite 
in  the  body  of  this  bug  was  described,  but  has  not  been 
confirmed.  Chagas  was  able  to  transmit  the  parasites  to 
monkeys  by  permitting  the  infected  bugs  to  bite  them. 


CHAPTER  XXI. 

KALA-AZAR  (BLACK  FEVER). 

LEISHMANIA  DONOVANI  (LAVERAN  AND  MESNIL). 

" KALA-AZAR,"  "  Dumdum  fever,"  "Febrile  tropical  spleno- 
megaly," "  Non-malarial  remittent  fever,"  is  a  peculiar,  fatal, 
infectious  disease  of  India,  Assam,  certain  parts  of  China,  the 
Malay  Archipelago,  North  Africa,  the  Soudan,  and  Arabia, 
caused  by  a  protozoan  micro-organism  known  as  Leishmania 
donovani,  and  characterized  by  irregular  fever,  great  enlarge- 
ment of  the  spleen,  anemia,  emaciation,  prostration,  not  in- 
frequent dysentery,  occasional  ulcerations  of  the  skin  and 
mucous  membranes,  and  sometimes  cancrum  oris. 

Because  of  its  protean  manifestations  the  disease  has  been 
given  many  names,  and  has  been  confused  with  the  various 
diseases  which  its  symptoms  may  resemble.  It  was  not 
until  1900  that  it  was  finally  differentiated  from  malarial 
fever  and  came  to  be  regarded  as  a  distinct  entity. 

In  1900  Leishman*  noticed  in  the  spleen  of  a  soldier  re- 
turned from  India  and  suffering  from  "  dumdum  fever  " — 
a  fever  acquired  at  Dumdum,  an  unhealthy  military  can- 
tonment not  far  from  Calcutta — certain  peculiar  bodies.  He 
reserved  publishing  the  observation  until  1903,  so  that  it 
appeared  almost  simultaneously  with  a  paper  upon  the  same 
subject  by  Donovan,  f  As  the  publications  came  from  men 
in  different  parts  of  the  world,  appeared  so  nearly  at  the*  same 
time,  and  showed  that  they  had  independently  arrived  at  the 
same  discovery,  the  parasite  they  described  became  known 
as  the  Leishman- Donovan  body.  For  a  long  time  its  nature 
was  not  known  and  its  proper  classification  impossible,  but 
after  it  had  been  carefully  studied  by  Rogers  J  Ross,§  and 

*"Brit.  Med.  Jour.,"  1903,  i,  1252. 

t  Ibid.,  1903,  n,  79. 

J  "Quarterly  Jour.  Microscopical  Society,"  XLVIII,  367;  "Brit.  Med. 
Jour.,"  1904,  i,  1249;  n,  645;  "Proceedings  of  the  Royal  Society,"  LXXVII, 
284. 

§  "Brit.  Med.  Jour.,"  1903,  n,  1401, 

566 


Leishmania  Donovan! 


567 


others,  and  its  developmental  forms  observed,  it  was  agreed 
that  it  belonged  in  a  new  genus  of  micro-organisms,  not  far 


•       *%        f9       •• 


%        !•      »- 


•,  .fl 

V-  '* 


•  f 


A* 

rSi^- 

» 


Fig.  195. — Evolution  of  the  parasite  of  kala-azar:  i  to  5.  Parasites 
of  kala-azar.  i,  Isolated  parasites  of  different  forms  in  the  spleen  and 
liver;  2,  division  forms  from  liver  and  bone-marrow;  3,  mononuclear 
spleen  cells  containing  the  parasites;  4,  group  of  parasites;  5,  phagocy- 
tosis of  a  parasite  by  a  polynuclear  leukocyte.  6  to  15.  Parasites  from 
cultures.  6,  First  changes  in  the  parasites.  The  protoplasm  has  in- 
creased in  bulk  and  the  nucleus  has  become  larger;  7,  further  increase 
in  size;  vacuolization  of  the  protoplasm;  8,  division  of  the  enlarged 
parasite;  9,  evolution  of  the  flagella;  10,  small  piriform  parasite  show- 
ing flagellum;  n,  further  development  and  division  of  the  parasite;  12, 
flagellated  trypanosoma-like  form;  13,  14,  flagellated  forms  dividing  by 
a  splitting  off  of  a  portion  of  the  protoplasm;  15,  narrow  flagellated 
parasites  which  have  arisen  by  the  type  of  division  shown  in  13  and  14. 
(From  Mense's  "  Handbuch,"  after  Leishman.) 


removed  from  the  trypanosomes,  and  eventually  Ross,  and 
then  Laveran  and  Mesnil,  honored  both  of  its  discoverers  by 


568  Kala-Azar 

calling  it  Leishmania  donovani,  which  name  has  been  gener- 
ally accepted. 

Morphology. — As  seen  in  a  drop  of  splenic  pulp  the  organ- 
ism is  a  minute  round  or  oval  intracellular  body  measuring 
2.5  by  3.5  (A.  When  properly  stained  with  polychrome 
methylene-blue  (Wright's,  Irishman's,  or  Jenner's  stains)  and 
examined  under  a  high  magnification,  it  is  found  that  the 
protoplasm  takes  a  pinkish  color  and  contains  two  well- 
defined  bright  red  bodies.  The  larger  of  these  is  ovoid  and  lies 
excentrically,  its  long  diameter  corresponding  to  the  long  di- 
ameter of  the  organism.  This  is  regarded  as  the  nucleus. 


Fig.  196. — Leishman-Donovan  bodies  from  the  spleen  of  a  case  of 
kala-azar.  X  about  1000.  (From  Seattle  and  Dickinson's  "A  Text- 
Book  of  General  Pathology,"  by  kind  permission  of  Rebman,  Limited, 
publishers.) 

The  second  body  is  smaller  and  of  bacillary  shape,  and  usu- 
ally lies  with  its  long  diameter  transverse  to  the  nucleus. 
This  is  looked  upon  as  a  blepharoplast.  It  stains  more  in- 
tensely than  the  nucleus.  In  addition  to  these  bodies  the 
protoplasm  may  contain  one  or  two  vacuoles. 

All  of  the  bodies  are  intracellular,  as  can  easily  be  deter- 
mined by  examining  sections  of  tissue,  but  in  smears  of  splenic 
pulp  the  cells  are  broken  and  many  free  bodies  may  appear. 
The  cells  in  which  they  occur  are  lymphocytes,  endothelial 
cells,  and  peculiar  large  cells  whose  histogenesis  is  obscure. 
They  are  rarely  to  be  found  in  polymorphonuclear  leukocytes, 
and  though  there  has  been  much  discussion  upon  this  point, 
probably  never  appear  in  the  red  blood-corpuscles. 


Cultivation 


569 


The  bodies  divide  by  binary  and  multiple  fission,  without 
recognizable  mitotic  changes.  When  multiple  fission  occurs, 
the  nucleus  divides  several  times  before  the  protoplasm 
breaks  up.  The  organism  is  not  motile  and  at  this  stage  has 
no  flagella. 

Cultivation. — The  organism  was  first  cultivated  artificially 
by  Rogers  in  citrated  splenic  juice  at  17°  to  24°  C.  It  can 
also  be  cultivated  in  the  blood-serum  agar  medium  used 
by  Novy,  MacNeal,  and  Hall  for  trypanosomes. 

Under  conditions  of  cultivation  the  appearance  of  the  or- 
ganism undergoes  a  complete  change.  It  enlarges,  the 
nucleus  increases  greatly  in  size,  and  a  pink  vacuole  appears 
near  the  blepharoplast.  In  the  course  of  twenty-four  to 


Fig.  197. — Leishmania  donovani.     Flagellated  forms  obtained  in  pure 
cultures  (Irishman) . 

forty-eight  hours  the  organism  elongates,  the  blepharoplast 
moves  to  one  end,  and  from  the  vacuole  near  it  a  flagellum  is 
developed,  and  the  organism  becomes  in  about  ninety-six 
hours  a  flagellate  protozoan  resembling  herpetomonas.  It 
now  measures  about  20  p  in  length  and  3  to  4  {i  in  breadth,  its 
whip  or  flagellum  measuring  about  3  /w  additional.  It  is  also 
motile  and,  like  the  trypanosomes,  swims  with  the  flagellum 
anteriorly.  There  is  no  undulating  membrane. 

This  may  be  regarded  as  the  perfect  or  adult  form  of  the 
organism.  It  multiplies  by  a  peculiar  mode  of  division  first 
observed  by  Leishman.  Chromatin  granules,  a  larger  and 
a  smaller,  appear  in  the  protoplasm  in  pairs,  after  which, 
through  unequal  longitudinal  cleavage,  long,  slender,  almost 
hair-like  individuals,  containing  one  of  the  pairs  of  chromatin 
granules,  are  separated.  These  were  serpentine  at  first,  but 


570  Kala-Azar 

later,  as  they  grew  larger,  a  flagellum  was  thrust  out  at  one 
end. 

Distribution. — The  Leishman-Donovan  body  is  widely 
distributed  throughout  the  body  of  the  patients  suffering 
from  kala-azar.  It  occurs  in  great  numbers  in  the  cells  of  the 
spleen,  of  the  liver,  of  the  bone-marrow,  and  in  the  ulcerations 
of  the  mucous  membranes  and  skin.  In  the  peripheral  blood 
they  are  few  and  only  in  the  leukocytes.  They  are  always 
intracellular,  or  when  in  the  circulating  blood  may  be  found 
in  indefinite  albuminous  masses,  probably  destroyed  cells. 
The  number  in  a  cell  varies  up  to  several  hundred,  such  great 
aggregations  only  being  found  in  the  peculiar  large  cells  of 
the  spleen. 

Lesions. — The  splenomegaly  is  the  most  striking  lesion. 
The  change  by  which  the  enlargement  is  effected  is  not  spe- 
cific. The  organ  is  not  essentially  changed  histologically,  but 
seems  to  be  merely  hyperplastic.  The  liver  is  enlarged,  but 
here,  again,  specific  changes  may  be  absent.  In  some  cases 
a  pallor  of  the  centers  of  the  lobules  may  depend  upon 
numbers  of  parasite-containing  cells,  partly  degenerated. 

The  yellow  bone-marrow  becomes  absorbed  and  red  tissue 
takes  its  place,  as  in  most  profound  anemias. 

Transmission. — Roger's  observation,  that  the  round 
bodies  grew  into  flagellate  bodies  at  temperatures  much  below 
that  of  the  human  body,  led  Manson  to  conjecture  that  the 
extra-human  phase  of  the  life  of  the  organism  took  place  at 
similar  low  temperatures  in  the  soil  or  in  water.  Patton* 
found  that  a  number  of  cases  sometimes  occurred  in  the  same 
house,  while  neighboring  houses  were  free,  and  thought  this 
suggested  that  a  domestic  insect  might  be  the  distributing 
host.  He  made  some  unconvincing  experiments  on  the  sub- 
ject. Rogers  tried  to  convict  the  bedbug,  but  also  failed. 
The  rarity  of  the  Leishmania  in  the  peripheral  blood  seems 
opposed  to  its  transmission  by  insects. 

On  the  other  hand,  the  organisms  leave  the  body  in  con- 
siderable numbers  in  the  dejecta  of  patients  suffering  from 
ulcerative  lesions  of  the  intestines  and  in  the  discharged  pus 
from  the  ulcerations  of  the  skin. 

However,  in  the  present  state  of  knowledge  there  can  be 
nothing  but  speculation  upon  the  subject.  The  disease  ap- 
pears not  to  be  transmissible  to  any  of  the  laboratory  animals, 

*  "Scientific  Memoirs  of  the  Government  in  India,"  1907,  No.  27. 


Infantile  Kala-Azar  571 

hence  great  difficulties  surround  all  investigation  of  the 
problem  of  transmission. 

Diagnosis. — The  anemia  of  kala-azar  is  usually  not  pro- 
found. The  erythrocytes  number  about  3,000,000  in  ordi- 
nary cases  and  the  hemoglobin  is  correspondingly  diminished. 
As  in  malaria,  there  is  leukopenia,  but  it  is  usually  more  severe, 
the  white  corpuscles  sometimes  being  as  few  as  600  to  650  per 
cubic  millimeter  of  blood.  The  enlargement  of  the  spleen 
and  liver  suggest  malaria. 

The  only  certain  way  to  make  a  diagnosis,  except  in  those 
rare  cases  where  one  has  the  good  fortune  to  find  occasional 
parasites  in  the  leukocytes  of  the  circulating  blood,  is  by 
splenic  culture.  A  large  hypodermic  needle  should  be  used, 
should  be  carefully  sterilized,  thrust  into  the  spleen,  and  a 
bit  of  splenic  pulp  secured  by  firmly  withdrawing  the  piston. 

Before  making  such  a  puncture,  leukemia  should  be  ex- 
cluded, lest  hemorrhage  occur. 


INFANTILE  KALA-AZAR. 
'  LEISHMANIA  INFANTUM 

Nicolle,*  while  in  Tunis,  observed  a  form  of  kala-azar  that 
was  peculiar  to  childhood  and  most  frequent  in  babies  of 
about  two  years  of  age.  Mesnil  has  identified  the  affection 
with  a  disease  known  as  "ponos"  in  Greece.  In  the  spleens 
of  such  patients  Nicolle  found  an  organism  that  was  not  dis- 
tinguishable either  by  microscopic  examination  or  by  culti- 
vation from  Leishmaniadonovani,  but,  finding  that  it  was  in- 
fectious for  dogs,  he  came  to  the  conclusion  that  it  was  a 
separate  species,  and  called  it  Leishmaniainfantum.  He  also 
found  that  the  dogs  in  Tunis  frequently  suffered  from  spon- 
taneous infection  from  this  parasite,  and  it  is  possible  that  the 
dogs  are  the  source  from  which  the  children  become  infected. 

Pianesef  found  infantile  kala-azar  in  Italy,  and  in  the 
children  suffering  from  it  he  was  able  to  find  the  Leishmania 
infantum. 

Further  experiments  with  this  parasite  by  Nicolle  and 
Comte  have  shown  that  in  the  form  in  which  it  occurs  in  the 
human  spleen  it  is  capable  of  infecting  monkeys,  and  Novy 
has  succeeded  in  cultivating  the  organism  and  infecting  dogs 

*  "Ann.  de  1'Inst.  Pasteur,"  1909,  xxm,  361,  441. 
f'Gaz.  Intern,  di  Medicin,"  1905,  vm,  8. 


572  Kala-Azar 

with  the  artificial  cultures  containing  the  flagellate  forms  of 
the  organism 

TROPICAL  ULCER. 
LEISHMANIA  FURUNCULOSA  (FIRTH). 

In  India,  northern  Africa,  southern  Russia,  parts  of  China, 
the  West  Indies,  South  America,  and,  indeed,  most  tropical 
countries,  a  peculiar  intractable  chronic  ulceration  is  occa- 
sionally observed,  and  is  variously  known  as  Tropical  ulcer, 
Oriental  sore,  Biscra  boil,  Biscra  button,  Aleppo  boil,  Delhi 


i 


Fig.  198. — Helcosoma  tropicum,  from  a  case  of  tropical  ulcer  ("Delhi 
sore")  smear  preparation  from  the  lesion  stained  with  Wright's  Roman- 
owsky  blood-staining  fluid.  The  ring-like  bodies,  with  white  central 
portions  and  containing  a  larger  and  a  smaller  dark  mass,  are  the  micro- 
organisms. The  dark  masses  in  the  bodies  are  stained  a  lilac  color, 
while  the  peripheral  portions  of  the  bodies,  in  typical  instances,  are 
stained  a  pale  robin's  egg  blue.  The  very  dark  masses  are  nuclei  of 
cells  of  the  lesion.  X  1500  approx.  (Wright).  (From  photograph  by 
Mr.  L.  S.  Brown.) 

boil,  Bagdad  boil,  and  Buton  d'Orient.  It  has  long  been 
known  as  a  specific  ulcerating  granuloma.  The  lesions,  which 
begin  as  red  spots,  develop  into  papules  which  become  covered 
with  a  scaly  crust  which  separates,  leaving  an  ulcer  upon  which 
a  new  crust  develops.  The  lesion  spreads  and  is  much  larger 
when  the  crust  again  separates.  A  purulent  discharge  is 
given  off  in  moderate  quantities  and  the  ulcer  becomes  deep 
and  perpendicularly  excavated.  It  lasts  for  months — some- 
times a  year  or  more — and  gradually  cicatrizes,  forming  a  con- 
tracting scar  that  is  quite  disfiguring  when  upon  the  face. 


Histoplasmosis  573 

The  lesions  may  be  single,  though  they  are  commonly  mul- 
tiple, as  many  as  twenty  sometimes  occurring  simultaneously. 
It  is  thought  that  recovery  is  followed  by  immunity. 

In  1885  Cunningham*  described  a  protozoan  organism 
found  in  the  tropical  ulcer,  the  observation  being  confirmed 
by  Firth,  f  who  called  the  bodies  Sporozoa  furunculosa. 
Later,  J.  H.  Wright  J  studied  a  case  of  tropical  ulcer  and  found 
bodies  precisely  like  the  Leishmania  donovani.  He  gave  it  the 
name  Helcosoma  tropicum.  The  great  similarity  to  the  other 
organisms  has  led  more  recent  writers  to  identify  it  with 
Leishmania,  but  as  it  induces  a  local  and  not  a  general  infec- 
tion like  kala-azar,  it  is  now  known  as  Leishmania  furunculosa. 

The  organism  has  not  been  cultivated.  It  can  undoubtedly 
be  transmitted  by  inoculation  from  human  being  to  human 
being,  and  Jackson  §  is  authority  for  the  statement  that  "  the 
Jews  of  Bagdad  recognized  that  tropical  ulcer  is  inoculable 
and  autoprotective  years  ago,  and  practiced  vaccination  of 
their  children  upon  some  portion  of  the  body  covered  by 
clothing,  in  order  that  their  faces  and  other  exposed  parts  of 
the  body  be  not  disfigured  by  the  ulcers  and  the  resultant 
scars." 

The  mode  of  transmission  is  not  known,  but  as  the  lesions 
usually  occur  where  the  body  surface  is  uncovered,  it  may 
be  that  flies  or  other  insects  are  concerned  in  the  transmission 
of  the  parasites. 

HISTOPLASMOSIS. 
HISTOPLASMA  CAPSULATUM  (DARLING). 

In  1906  Darling,  ||  working  at  the  Isthmus  of  Panama,  ob- 
served certain  cases  presenting  pyrexia,  anemia,  leukopenia, 
splenomegaly,  and  emaciation,  and  bearing  a  close  resem- 
blance to  kala-azar.  The  disease  was  quite  chronic,  and  it 
terminated  fatally.  When  examined  at  autopsy,  these  cases 
showed  necrosis  with  cirrhosis  of  the  liver,  splenomegaly, 
pseudogranulomata  of  the  lungs,  small  and  large  intestines, 

*"  Scientific  Memoirs  by  Medical  Officers  of  the  Army  in  India," 
1884,  i. 

t  "British  Med.  Journal,"  1891,  Jan.  10,  p.  60. 

t  ''Jour,  of  Med.  Research,"  x,  1904,  472. 

§  "Tropical  Medicine,"  Phila.,  P.  Blakiston's  Son  &  Co.,  1907,  p. 
478. 

||  "Jour.  Amer.  Med.  Assoc.,"  1906,  XLVI,  1283;  "Archiv.  f.  Int.  Med." 
1908,  n,  107;  "Jour.  Exp.  Med.,"  1909,  xi,  515. 


574 


Kala-Azar 


ulceration  of  the  intestines,  and  necrosis  of  the  lymph-nodes 
draining  the  injected  viscera.  The  lesions  seemed  to  depend 
upon  the  invasion  of  the  endothelial  cells  of  the  smaller 
lymph-  and  blood-vessels  by  enormous  numbers  of  a  small 
encapsulated  micro-organism. 

The  organism  is  small,  round  or  oval  in  shape,  and  meas- 
ures  i  to  4  ft  in  diameter.      It  possesses  a  polymorphous, 


Fig.  199. — Histoplasma  capsulatum.  Mononuclear  cells  from  the 
lung  containing  many  parasites  (Darling).  (Samuel  T.  Darling  in 
"Journal  of  Experimental  Medicine.") 

chromatin  nucleus,  basophilic  cytoplasm,  and  achromatic 
spaces  all  enclosed  within  an  achromatic  refractile  capsule. 
The  micro-organism  differs  from  the  Leishman-Donovan 
body  of  kala-azar  in  the  form  and  arrangement  of  its  chro- 
matin nucleus  and  in  not  possessing  a  chromatin  rod.  The 
distribution  of  the  parasite  in  the  body  is  accomplished  by 
the  invasion  of  the  contiguous  endothelial  cells  of  the  smaller 


Histoplasmosis  575 

blood-  and  lymph-vessels  and  capillaries,  and  by  the  infec- 
tion of  distant  regions  by  the  dislodgment  of  infected  endo- 
thelial  cells  and  their  transportation  thither  by  the  blood-  and 
lymph -stream.  Thus  the  skin,  intestinal,  and  pulmonary 
nodules  may  be  due  to  secondary  distribution  of  the  parasite. 
The  micro-organism  apparently  lives  for  a  considerable 
period  of  time  iin  the  tissues,  because  in  the  older  areas  of 
necrosis  there  are  myriads  of  parasites  all  staining  well. 

The  mode  of  infection  and  portal  of  entry  are  unknown. 
The  parasite  has  neither  been  cultivated  nor  transmitted  by 
inoculation. 

Believing  it  to  be  a  new  parasite,  Darling  has  suggested 
that  it  be  called  Histoplasma  capsulatum. 


CHAPTER   XXII 

YELLOW  FEVER. 

THE  bacteriology  of  yellow  fever  has  been  studied  by 
Domingos  Freire,*  Carmona  y  Valle,f  Sternberg,J  Havel- 
burg^  and  Sanarelli,||  but  all  of  their  work  has  been  shown 
to  be  incorrect  by  the  interesting  researches  and  very  con- 
clusive results  of  Finlay,**  Carter,  ff  Reed,  Carroll,  Lazear, 
and  Agramonte,  J  {  and  Reed  and  Carroll,  §§  which  have 
proved  the  mosquito  to  be  the  definitive  host  of  an  invis- 
ible micro-organism. 

Reed,  Carroll,  Lazear,  and  Agramonte,  ||||  constituting  a 
Board  of  Medical  Officers  "for  the  purpose  of  pursuing  scien- 
tific investigations  with  reference  to  the  acute  infectious  dis- 
eases prevalent  on  the  island  of  Cuba,"  began  their  work  in 
1900,  at  Havana,  by  a  careful  investigation  of  the  relationship 
of  Bacillus  icteroides  to  yellow  fever.  By  a  most  careful  tech- 
nic  they  withdrew  and  examined  the  blood  from  the  veins  of 
the  elbow  of  1 8  cases  of  yellow  fever,  making  48  separate  ex- 
aminations on  different  days  of  the  disease,  and  preparing 
115  bouillon  cultures  and  18  agar  plates,  every  examination 
being  negative  so  far  as  Bacillus  icteroides  was  concerned. 

*"  Doctrine  microbienne  de  la  fievre  jaune  et  ses  inoculation  pre- 
ventives," Rio  Janeiro,  1885. 

f  "Lemons  sur  1'etiologie  et  la  prophylaxie  de  la  fievre  jaune,"  Mexico, 
1885. 

t  "Report  on  the  Etiology  and  Prevention  of  Yellow  Fever,"  Wash- 
ington, 1891;  "Report  on  the  Prevention  of  Yellow  Fever  by  Inocula- 
tion," Washington,  1888. 

§  "Ann.  de  1'Inst.  Pasteur,"  1897. 

||  "Brit.  Med.  Jour.,"  July  3,  1897;  "Ann.  de  1'Inst.  Pasteur," 
June,  Sept.,  and  Oct.,  1897. 

**  "Amer.  Jour.  Med.  Sci.,"  1891,  vol.  en,  p.  264;  "Ann.  de  la  Real 
Academia,"  vol.  xvm,  1881,  pp.  147-169;  "Jour.  Amer.  Med.  Assoc.," 
vol.  xxxvm,  April  19,  1902,  p.  993. 

ft  "New  Orleans  Med.  Jour.,"  May,  1890. 

U  "Phila.  Med.  Jour.,"  Oct.  27,  1900;  "Public  Health,"  vol.  xxvi, 
1900,  p.  23. 

§§  "Public  Health,"  vol.  xxvn,  1901,  p.  113. 

Ill)  "Phila.  Med.  Jour.,"  Oct.  27,  1900. 

576 


Mosquitoes  and  Yellow  Fever  577 

They  were  entirely  unable  to  confirm  the  findings  of  Wasdin 
and  Geddings,*  that  Bacillus  icteroides  was  present  in  blood 
obtained  from  the  ear  in  13  out  of  14  cases,  and  concluded 
that  both  Sanarelli  and  Wasden  and  Geddings  were  mistaken 
in  their  deductions. 

In  lieu  of  the  remarkably  interesting  discoveries  of  Ronald 
Ross  concerning  the  relation  of  the  mosquito  to  malarial  in- 
fection, the  commissioners,  remembering  the  theory  of 
Finlay,t  who  in  1881  published  an  experimental  research 
showing  that  mosquitoes  spread  the  infection  of  yellow  fever, 
and  the  interesting  and  valuable  observations  of  Carter  J 
upon  the  interval  between  infecting  and  secondary  cases  of 
yellow  fever,  turned  their  attention  to  the  mosquito.  Secur- 
ing mosquitoes  from  Finlay  and  continuing  the  work  where 
he  had  left  it,  they  found  that  when  mosquitoes  (Stegomyia 
fasciata  sen  calopus}  were  permitted  to  bite  patients  suffering 
from  yellow  fever,  after  an  interval  of  about  twelve  days 
they  became  able  to  impart  yellow  fever  with  their  bites. 
This  infectious  character  of  the  bite,  having  once  developed, 
seems  to  remain  throughout  the  subsequent  life  of  the 
insect.  So  far  as  it  was  possible  to  determine,  only  one 
species  of  mosquito,  Stegomyia  calopus,  served  as  a  host 
for  the  parasite  whose  cycles  of  development  in  the  mosquito 
and  in  man  must  explain  the  symptomatology  of  yellow 
fever. 

In  order  to  establish  these  observations,  experimental 
inoculations  were  made  upon  human  beings  in  sufficient 
number  to  prove  their  accuracy.  Unfortunately,  Dr. 
Lazear,  one  of  the  victims  of  the  experiment,  lost  his  life 
from  an  attack  of  yellow  fever. 

Reed,  Carroll,  and  Agramonte§  came  to  the  following 
conclusions : 

1 .  The  mosquito  C.  fasciatus  (Stegomyia  calopus)  serves 
as  the  intermediate  host  of  the  yellow  fever  parasite. 

2.  Yellow  fever  is  transmitted  to  the  non-immune  indi- 
vidual by  means  of  the  bite  of  the  mosquito  that  has  pre- 
viously fed  on  the  blood  of  those  sick  with  the  disease. 

*  "  Report  of  the  Commission  of  Medical  Officers  Detailed  by  the 
Authority  of  the  President  to  Investigate  the  Cause  of  Yellow  Fever," 
Washington,  D.  C.,  1899. 

f  "Annales  de  la  Real  Academia,"  vol.  xvm,  1881,  pp.  147-169. 

t  "New  Orleans  Med.  Jour.,"  May,  1900. 

\  Pan-American  Medical  Congress,  Havana,  Cuba,  Feb.  4-7,  1901; 
Sanitary  Department,  Cuba,  series  3,  1902. 

37 


578 


Yellow  Fever 


3.  An  interval  of  about  twelve  days  or  more  after  con- 
tamination appears  to  be  necessary  before  the  mosquito  is 
capable  of  conveying  the  infection. 


Fig.  200. — Stegomyia  fasciata  (Stegomyia  calopus):  a,  female;  b,  male 
(after  Carroll) . 

4.  The  bite  of  the  mosquito  at  an  earlier  period  after 
contamination  does  not  appear  to  confer  any  immunity 
against  a  subsequent  attack. 


Mosquitoes  and  Yellow  Fever  579 

5.  Yellow  fever  can  be  experimentally  produced  by  the 
subcutaneous   injection   of   blood   taken   from   the   general 
circulation  during  the  first  and  second  days  of  the  disease. 

6.  An  attack  of  yellow  fever  produced  by  the  bite  of  a 
mosquito  confers  immunity  against  the  subsequent  injection 
of  the  blood  of  an  individual  suffering  from  the  non-experi- 
mental form  of  the  disease. 

7.  The  period  of  incubation  in  13  cases  of  experimental 
yellow  fever  has  varied  from  forty -one  hours  to  five  days  and 
seventeen  hours. 

8.  Yellow  fever  is  not  conveyed  by  fomites,  and  hence 
disinfection  of  articles  of  clothing,  bedding,  or  merchandise, 
supposedly  contaminated  by  contact  with  those  sick  with  the 
disease,  is  unnecessary. 

9.  A  house  may  be  said  to  be  infected  with  yellow  fever 
only  when  there  are  present  within  its  walls  contaminated 
mosquitoes  capable  of  conveying  the  parasite  of  this  disease. 

10.  The  spread  of  yellow  fever  can  be  most  effectually 
controlled  by  measures  directed  to  the  destruction  of  mos- 
quitoes and  the  protection  of  the  sick  against  the  bites  of 
these  insects. 

11.  While  the  mode  of  propagation  of  yellow  fever  has 
now  been  definitely  determined,  the  specific  cause  of  the 
disease  remains  to  be  discovered. 

The  probability  that  Bacillus  icteroides  is  the  specific 
cause  and  is  transmitted  by  the  mosquito  is  so  slight  that  it 
need  scarcely  be  considered.  All  analogy  points  to  the 
organism  being  an  animal  parasite  similar  to  that  of  malarial 
fever. 

With  this  positive  information  before  us,  the  prophylaxis 
of  yellow  fever  and  the  prevention  of  epidemics  of  the  disease 
where  sporadic  cases  occur  becomes  very  simple  and  may  be 
expressed  in  the  following  rules: 

1.  Whenever  yellow  fever  is  likely  to  occur,  the  breeding 
places  of  mosquitoes  should  be  destroyed  by  drainage.     Cis- 
terns and  other  necessary  collections  of  standing  water  should 
be  covered  or  secured. 

2.  Houses  should  have  the  windows  and  doors  screened 
and  the  inhabitants  should  use  bed  nets. 

3.  So  soon  as  a  case  of  fever  appears  it  should  be  removed  in 
a  mosquito-proof  ambulance  to  a  mosquito-proof  apartment 
in  a  well-screened  hospital  ward  and  kept  there  until  con- 
valescent. 


580  Yellow  Fever 

4.  The  premises  where  such  a  case  has  occurred  should  be 
fumigated  by  burning  pyre  thrum  powder  (i  pound  per  1000 
cubic  feet)  to  stun  the  mosquitoes,  which  fall  to  the  floor  and 
must  afterward  be  swept  up  and  destroyed. 

By  these  means  Major  W.  C.  Gorgas,*  without  expensive 
disinfection  and  without  regard  for  fomites,  has  virtually 
exterminated  yellow  fever  from  Havana  and  from  the  Canal 
Zone,  Panama,  where  it  was  for  many  years  endemic. 

A  practical  point  connected  with  the  screens  is  given  in  the 
work  of  Rosenau,  Parker,  Francis,  and  Beyer,  |  who  found  that 
to  be  effective  the  screens  must  have  20  strands  or  19  meshes 
to  the  inch.  If  coarser  than  this  the  stegomyia  mosquitoes 
can  pass  through. 

Reed  and  Carroll  {  were  the  first  to  filter  the  blood  of  yellow 
fever  patients  and  prove  that  after  it  had  passed  through  a 
Berkefeld  filter  that  kept  back  Staphylococcus  aureus,  it 
still  remained  infective  and  capable  of  producing  yellow  fever 
in  non-immune  human  beings. 

This  subject  was  further  investigated  by  Rosenau,  Parker, 
Francis  and  Beyer,  §  who  found  that  the  virus  was  even  smaller 
than  the  first  experiment  would  suggest,  as  it  not  only  passed 
through  the  Berkefeld  filter,  but  also  through  the  Pasteur- 
Chamberland  filter.  The  filtrates  always  remained  sterile 
when  added  to  culture-media. 

The  virus  has  not  been  artificially  cultivated. 

Prophylaxis. — Guiteras||  has  studied  the  effect  of  inten- 
tionally permitting  non-immunes  who  are  to  be  exposed  to  the 
disease  to  be  experimentally  infected  by  being  bitten  by  in- 
fected mosquitoes,  after  which  they  are  at  once  carefully 
treated.  His  first  conclusion  was  that  "the  intentional  inoc- 
ulation gives  the  patient  a  better  chance  of  recovery,"  but  the 
danger  of  death  from  the  experimental  injection  was  later 
shown  to  be  so  great  that  it  had  to  be  abandoned. 

*  International  Sanitary  Congress  held  at  Havana,  Cuba,  Feb.  16, 
1902;  Sanitary  Department,  Havana,  series  4. 

f  Report  of  Working  Party  No.  2,  Yellow  Fever  Institute,  Bull.  14, 
May,  1904. 

|  "Am.  Med.,"  Feb.  22,  1902. 

§  "Bull.  No.  14,  U.S.  Public  Health  and  Marine  Hospital  Service," 
Washington,  D.  C.,  May,  1904. 

||  "Revista  de  Medicina  Tropical,"  Havana,  Cuba,  1902. 


CHAPTER   XXIII.  • 

PLAGUE. 

BACILLUS  PESTIS  (YERSIN,  KITASATO). 

General  Characteristics. — A  minute,  pleomorphous,  diplococcoid 
and  elongate,  sometimes  branched,  non-motile,  non-flagellated,  non- 
sporogenous,  non-chromogenic,  aerobic  and  optionally  anaerobic, 
pathogenic  organism,  specific  for  bubonic  plague,  easily  cultivated 
artificially,  and  susceptible  of  staining  by  ordinary  methods,  but  not 
by  Gram's  method. 

Plague,  bubonic  plague,  pest,  black  plague,  "  black  death," 
or  malignant  polyadenitis  is  an  acute  epidemic  infectious 
febrile  disease  of  an  intensely  fatal  nature,  characterized  by 
inflammatory  enlargement  and  softening  of  the  lymphatic 
glands,  marked  pulmonary,  cerebral,  and  vascular  disturb- 
ance, and  the  presence  of  the  specific  bacillus  in  the  lymphatic 
glands  and  blood. 

The  history  of  plague  is  so  full  of  interest  that  many  ref- 
erences to  it  appear  in  popular  literature.  The  student  can 
scarcely  find  more  profitable  reading  than  the  "History  of  the 
Plague  Year  in  London,"  by  DeFoe,  and  readers  of  Boccacio 
will  remember  that  it  was  the  plague  epidemic  then  raging  in 
Florence  that  led  to  the  isolation  of  the  group  of  young 
people  by  whom  the  charming  stories  of  the  Decameron  were 
told. 

During  the  reign  of  the  Emperor  Justinian  the  plague  is 
said  to  have  carried  off  nearly  half  of  the  population  of  the 
Roman  Empire.  In  the  fourteenth  century  it  is  said  to  have 
destroyed  nearly  twenty-five  millions  of  the  population  of 
Europe.  Epidemics  of  less  severity  but  attended  with  great 
mortality  appeared  in  the  sixteenth,  seventeenth,  and  eight- 
eenth centuries.  In  1894  an  epidemic  broke  out  in  the 
western  Chinese  province  of  Yunnan  and  reached  Canton  in 
January,  1894,  thus  escaping  from  its  endemic  center  and 
began  to  spread.  It  can  be  traced  from  Canton  to  Hong- 
kong. In  1895  it  appeared  also  in  Amoy,  Macao,  and 
Foochoo.  In  1896  it  had  reached  Bombay  and  reappeared  in 
Hongkong.  In  1897  Bombay,  the  Madras  Presidency,  the 

581 


582 


Plague 


Punjab,  and  Madras  were  visited.  In  1898  the  disease  spread 
greatly  throughout  India  and  into  Turkestan,  and  by  sea  went 
to  Madagascar  and  Mauritius.  In  1899  it  extended  still 
more  widely  in  India  and  China,  Japan  and  Formosa,  and 
succeeded  in  disseminating  as  widely  as  the  Hawaiian  Islands 
and  New  Caledonia  on  the  east,  Portugal,  Russia,  and 
Austria  on  the  west,  and  Brazil  and  Paraguay  on  the  south. 
In  1900  it  had  spread  to  nearly  every  part  of  the  world.  In 
these  places  when  sanitary  measures  could  not  be  carried  into 
effect  the  people  died  in  great  numbers — thus  in  India  in 
1901  there  were  362,000  cases  and  278,000  deaths.  Where 


•I 


Fig.  201. — Axillary  bubo.  (Reproduced  from  Simpson's  "A  Treatise 
on  Plague,"  1905,  by  kind  permission  of  the  Cambridge  University 
Press.) 

the  precautions  were  possible  and  co-operation  between  the 
people  and  the  authorities  could  be  brought  about,  as  in  New 
York,  San  Francisco,  and  other  North  American  and  Euro- 
pean ports,  the  disease  remained  confined  pretty  well  within 
limits  and  did  not  spread.  An  interesting  account  of  "The 
Present  Pandemic  of  Plague"  by  J.  M.  Eager,  was  published 
in  1908  in  Washington,  D.  C.,  by  the  U.  S.  Public  Health  and 
Marine  Hospital  Service. 

Plague  is  an  extremely  fatal  affection,  whose  ravages  in  the 
hospital  at  Hongkong,  in  which  Yersin  made  his  original 
observations,  carried  off  95  per  cent,  of  the  cases.  The 
death-rate  varies  in  different  epidemics  from  50  to  90  per 


Plague  583 

cent.  In  the  epidemic  at  Hongkong  in  1894  the  death-rate 
was  93.4  per  cent,  for  Chinese,  77  per  cent,  for  Indians,  60 
per  cent,  for  Japanese,  100  per  cent,  for  Eurasians,  and  18.2 
per  cent,  for  Europeans.  It  affects  both  men  and  animals, 
and  is  characterized  by  sudden  onset,  high  fever,  prostration, 
delirium,  and  the  occurrence  of  exceedingly  painful  lymphatic 
swellings — buboes — affecting  chiefly  the  inguinal  glands, 
though  not  infrequently  the  axillary,  and  sometimes  the  cer- 
vical, glands.  Death  comes  on  in  severe  cases  in  forty-eight 
hours.  The  pneumonic  form  is  most  rapidly  fatal.  The 
longer  the  duration  of  the  disease,  the  better  the  prognosis. 
Autopsy  in  fatal  cases  reveals  the  characteristic  enlargement 
of  the  lymphatic  glands,  whose  contents  are  soft  and  some- 
times purulent. 

Wyman,*  in  his  very  instructive  pamphlet,  "The  Bu- 
bonic Plague,"  finds  it  convenient  to  divide  plague  into  (a) 
bubonic  or  ganglionic,  (6)  septicemic,  and  (c)  pneumonic 
forms.  Of  these,  the  bubonic  form  is  most  frequent  and  the 
pneumonic  form  most  fatal. 

The  infection  usually  takes  place  through  some  peripheral 
lesion,  but  may  occur  by  inhalation  of  the  specific  organ- 
isms. 

The  bacillus  of  bubonic  plague  was  independently  dis- 
covered by  Yersinj  and  KitasatoJ  in  the  summer  of  1894, 
during  an  epidemic  of  the  plague  then  raging  at  Hongkong. 
There  seems  to  be  little  doubt  but  that  the  micro-organisms 
described  by  the  two  observers  are  identical. 

Ogata  §  states  that  while  Kitasato  found  the  bacillus  in  the 
blood  of  cadavers,  Yersin  seldom  found  it  in  the  blood,  but 
always  in  the  enlarged  lymphatic  glands;  that  Kitasato's 
bacillus  retains  the  color  when  stained  by  Gram's  method; 
Yersin's  does  not;  that  Kitasato's  bacillus  is  motile;  Yersin's 
non-motile;  that  the  colonies  of  Kitasato's  bacillus,  when 
grown  upon  agar,  are  round,  irregular,  grayish  white,  with 
a  bluish  tint,  and  resemble  glass-wool  when  slightly  mag- 
nified; those  of  Yersin's  bacillus,  white  and  transparent, 
with  iridescent  edges.  Ogata,  in  his  investigations,  found 
that  the  bacillus  corresponded  with  the  description  of 

*  Government  Printing  Office,  Washington,  D.  C.,  1900. 

t  "Ann.  de  1'Inst.  Pasteur,"  1894,  9. 

t  Preliminary  notice  to  the  bacillus  of  bubonic  plague,  Hongkong, 
July  7,  1894- 

§"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Sept.  6,  1897,  Bd.  xxn, 
Nos.  6  and  7,  p.  170. 


584  Plague 

Yersin  rather  than  that  of  Kitasato,  and  it  is  certain  that 
the  description  given  by  Yersin  is  the  more  correct  of  the 
two. 

In  the  "JaPan  Times,"  Tokio,  November  28,  1899,  Kita- 
sato explains  that,  his  investigations  being  made  upon 
cadavers  that  were  partly  putrefied,  he  was  led  to  believe 
that  the  bacillus  first  invaded  the  blood.  Later  studies 
upon  living  subjects  showed  him  the  error  of  this  view  and 
the  correctness  of  Yer sin's  observation  that  the  bacilli  first 
multiply  in  the  lymphatics. 

Both  Kitasato  and  Yersin  showed  that  in  blood  drawn 
from  the  finger-tips  and  in  the  softened  contents  of  the 
glands  the  bacillus  may  be  demonstrable. 


Fig.  202. — Bacillus  of  bubonic  plague  (Yersin). 

Morphology. — The  bacillus  is  quite  variable.  Usually  it 
is  short  and  thick — a  "coco-bacillus,"  as  some  call  it — 
with  rounded  ends.  Its  size  is  small  (1.5  to  2  ^  in  length) 
and  0.5  to  0.75  ^  in  breadth.  It  not  infrequently  occurs  in 
chains  of  four  or  six  or  even  more,  and  is  occasionally  en- 
capsulated. It  shows  active  Brownian  movements,  which 
probably  led  Kitasato  to  consider  it  motile.  Yersin  did  not 
regard  it  as  motile,  and  was  correct.  Gordon*  claims  that 
some  of  the  bacilli  have  flagella.  No  spores  are  formed. 

Staining. — It  stains  by  the  usual  methods;  not  by 
Gram's  method.  When  stained,  the  organism  rarely 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  June  24,  1897,  Bd.  xxi,  Nos. 
20  and  2 1 . 


Cultivation  585 

appears  uniformly  colored,  being  darker  at  the  ends  than 
at  the  center,  so  as  to  resemble  a  dumb-bell  or  diplococcus. 
The  bacilli  sometimes  appears  vacuolated,  and  nearly  all 
cultures  show  a  variety  of  involution  forms.  Kitasato  has 
compared  the  general  appearance  of  the  bacillus  to  that  of 
chicken-cholera. 

Involution  forms  on  partly  desiccated  agar-agar  not 
containing  glycerin  are  said  by  Haffkine  to  be  characteris- 
tic. The  microbes  swell  and  form  large,  round,  oval,  pea- 
shaped,  spindle-shaped  or  biscuit-like  bodies  which  may  at- 
tain twenty  times  the  normal  size,  and  gradually  lose  the 


Fig.  203. — Bacilli  of  plague  and  phagocytes,  from  human  lymphatic 
gland.      X  800  (Aoyama). 

ability  to  take  the  stain.  Such  involution  forms  are  not 
seen  in  liquid  culture. 

Cultivation. — Pure  cultures  may  be  from  the  blood  or 
from  the  softened  contents  of  the  buboes,  and  develop  well 
upon  artificial  media.  The  optimum  temperature  is  about 
30°  C.  The  extremes  at  which  growth  occurs  are  20°  and 
38°  C. 

Bouillon. — In  bouillon  a  diffuse  cloudiness  was  ob- 
served by  Kitasato,  though  Yersin  observed  that  the  cul- 
tures resembled  erysipelas  cocci,  and  contained  zooglea 
attached  to  the  sides  and  at  the  bottom  of  the  tube  of 
nearly  clear  fluid. 


586  Plague 

Haffkine*  found  that  when  an  inoculated  bouillon  cul- 
ture is  allowed  to  stand  perfectly  at  rest,  on  a  firm  shelf 


Fig.  204. — Bacillus  pestis.  Highly  virulent  culture  forty-eight 
hours  old,  from  the  spleen  of  a  rat.  Unstained  preparation  (Kolle  and 
Wassermann). 

or  table,  a  characteristic  appearance  develops.  In  from 
twenty-four  to  forty-eight  hours,  the  liquid  remaining  limpid, 
flakes  appear  underneath  the  surface,  forming  little  islands 


Fig.  205. — Bacillus  pestis.  Involution  forms  from  a  pure  cul- 
ture on  3  per  cent,  sodium  chlorid  agar-agar.  Methylene-blue  (Kolle 
and  Wassermann). 

of  growth,   which  in  the    next  twenty-four    to  forty-eight 
hours  grow  into  a  jungle  of  long  stalactite-like  masses,  the 

*  "Brit.  Med.  Jour.,"  June  12,  1897,  p.  1461. 


Cultivation  587 

liquid  remaining  clear.  In  from  four  to  six  days  these 
islands  become  still  more  compact.  If  the  vessels  be  dis- 
turbed, they  fall  like  snow  and  are  deposited  at  the  bottom, 
leaving  the  liquid  clear. 

Colonies. — Upon  gelatin  plates  at  22°  C.  the  colonies  may 
be  observed  in  twenty-four  hours  by  the  naked  eye.  They 
are  pure  white  or  yellowish  white,  spheric  when  deep  in  the 
gelatin,  flat  when  upon  the  surface,  and  are  about  the  size 
of  a  pin's  head.  The  gelatin  is  not  liquefied.  Upon  micro- 
scopic examination  the  borders  of  the  colonies  are  found  to 


Fig.  206. — Stalactite  growth  of  bacillus  pestis  in  bouillon.  (Repro- 
duced from  Simpson's  ''A  Treatise  on  Plague,"  1905,  by  kind  permission 
of  the  Cambridge  University  Press.) 

be  sharply  defined.  The  contents  become  more  granular  as 
the  age  increases.  The  superficial  colonies  are  occasionally 
surrounded  by  a  fine,  semi-transparent  zone. 

Klein*  says  that  the  colonies  develop  quite  readily  upon 
gelatin  made  from  beef  bouillon  (not  infusion),  appearing 
in  twenty-four  hours,  at  20°  C.,  as  small,  gray,  irregularly 
rounded  dots.  Magnification  shows  the  colonies  to  be 
serrated  at  the  edges  and  made  up  of  short,  oval,  some- 
times double  bacilli.  Some  colonies  contrast  markedly 
with  their  neighbors  in  that  they  are  large,  round,  or  oval, 
and  consist  of  longer  or  shorter,  straight  or  looped  threads 
of  bacilli.  The  appearance  was  much  like  that  of  the  young 

*"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  July  10,  1897,  xxi,  Nos.  24 
and  25. 


588  Plague 

colonies  of  Proteus  vulgaris.  At  first  these  were  regarded 
as  contaminations,  but  later  he  was  led  to  believe  that  their 
occurrence  was  characteristic  of  the  plague  bacillus.  The 
peculiarities  of  these  colonies  cannot  be  recognized  after 
forty-eight  hours. 

Gelatin  Punctures. — In  gelatin  puncture  cultures  the 
development  is  scant.  The  medium  is  not  liquefied;  the 
growth  takes  place  in  the  form  of  a  fine  duct,  little  points 
being  seen  on  the  surface  and  in  the  line  of  puncture.  Some- 
times fine  filaments  project  into  the  gelatin  from  the  central 
puncture. 

Abel  found  the  best  culture-medium  to  be  2  per  cent,  alka- 
line peptone  solution  containing  i  or  2  per  cent,  of  gelatin, 
as  recommended  by  Yersin  and  Wilson. 

Agar-agar. — Upon  agar-agar  the  bacilli  grow  freely,  but 
slowly,  the  colonies  being  whitish  in  color,  with  a  bluish  tint 
by  reflected  light,  and  first  appearing  to  the  naked  eye  when 
cultivated  from  the  blood  of  an  infected  animal  after  about 
thirty-six  hours'  incubation  at  37°  C.  Under  the  micro- 
scope they  appear  moist,  with  rounded  uneven  edges. 
The  small  colonies  are  said  to  resemble  tufts  of  glass-wool. 
Microscopic  examination  of  the  agar-agar  culture  shows  the 
presence  of  chains  resembling  streptococci. 

Upon  glycerin-agar  the  development  of  the  colonies  is 
slower,  though  in  the  end  the  colonies  attain  a  larger  size 
than  those  grown  upon  plain  agar. 

Hankin  and  Leumann*  recommended  for  the  differential 
diagnosis  of  the  plague  bacillus  a  culture-medium  prepared 
by  the  addition  of  2.5  to  3.5  per  cent,  of  salt  to  ordinary 
culture  agar-agar.  When  transplanted  from  ordinary  agar- 
agar  to  the  salt  agar-agar,  the  involution  forms  so  charac- 
teristic of  the  bacillus  are  formed  with  exceptional  rapidity. 
In  bouillon  containing  this  high  percentage  of  salt  the  stalac- 
tite formation  is  beautiful  and  characteristic. 

Blood-serum. — Upon  blood-serum,  growth,  at  the  tem- 
perature of  the  incubator,  is  luxuriant  and  forms  a  moist 
layer,  of  yellowish-gray  color,  unaccompanied  by  lique- 
faction of  the  serum. 

Potato. — Upon  potato  no  growth  occurs  at  ordinary 
temperatures.  When  the  potato  is  stood  in  the  incubator 
for  a  few  days  a  scanty,  dry,  whitish  layer  develops. 

*"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Oct.,  1897,  Bd.  xxn,  Nos. 
16  and  17,  p.  438. 


Metabolism  589 

Vital  Resistance. — Kitasato  found  that  the  plague 
bacillus  did  not  seem  able  to  withstand  desiccation  longer 
than  four  days;  but  Rappaport*  found  that  they  remained 
alive  when  kept  dry  upon  woolen  threads  at  20°  C.  for 
twenty-three  days,  and  Yersin  found  that  although  it  could 
be  secured  from  the  soil  beneath  an  infected  house  at  a  depth 
of  4  to  5  cm.,  the  virulence  of  such  bacilli  was  lost. 

Kitasato  found  that  the  bacillus  was  killed  by  two  hours' 
exposure  to  0.5  per  cent,  carbolic  acid,  and  also  by  exposure 
to  a  temperature  of  80°  C.  for  five  minutes.  Ogata  found 
the  bacillus  instantly  killed  by  5  per  cent,  carbolic  acid,  and 
in  fifteen  minutes  by  0.5  per  cent,  carbolic  acid.  In  o.i  per 
cent,  sublimate  solution  it  is  killed  in  five  minutes. 

According  to  Wyman,  the  bacillus  is  killed  by  exposure 
to  55°  C.  for  ten  minutes.  The  German  Plague  Commission 
found  that  the  bacilli  were  killed  by  exposure  to  direct  sun- 
light for  three  or  four  hours;  and  Bowhillf  found  that  they 
are  killed  by  drying  at  ordinary  room  temperatures  in  about 
four  days. 

Wilson  I  found  the  thermal  death-point  of  the  organism 
one  or  two  degrees  higher  than  that  of  the  majority  of 
non-sporulating  pathogenic  bacteria,  and  that  the  influ- 
ence of  sunlight  and  desiccation  cannot  be  relied  upon  to 
destroy  it. 

Rosenaui  found  temperature  the  most  important  factor, 
as  it  dies  quickly  when  kept  dry  at  37°  C.,  but  remains  alive 
for  months  when  kept  dry  at  19°  C.  Sunlight  kills  it  in  a 
few  hours.  A  temperature  of  70°  C.  is  invariably  fatal  in 
a  short  time. 

Metabolism. — The  bacillus  develops  under  conditions  of 
aerobiosis  and  anaerobiosis.  In  glucose-containing  media 
it  does  not  form  gas.  No  indol  is  formed.  Ordinarily  the 
culture-medium  is  acidified,  the  acid  reaction  persisting  for 
three  weeks  or  more. 

Ghon,||  Wernicke,**  and  others  who  have  studied  the  toxic 

*  Quoted  by  Wyman. 

f  "Manual  of  Bacteriological  Technique  and  Special  Bacteriology," 
1899,  p.  197. 

t  "Journal  of  Medical  Research,"  vol.  vi,  No.  i,  p.  53,  July,  1901. 
§  Bulletin  No.  4  of  the  Hygienic  Laboratory  of  the  U.  S.  Marine 
Hospital  Service,  1901. 

||  Wien,  1898. 
**  "Centralbl.  f.  Bakt.,"  etc.,  1898,  xxiv. 


590  Plague 

products  of  the  bacillus  all  incline  to  the  belief  that  it  forms 
only  endotoxin. 

Kossee  and  Overbeck,*  however,  believe  that  there  is,  in 
addition,  a  soluble  exotoxin  that  is  of  importance. 

Experimental  Infection. — Mice,  rats,  guinea-pigs,  rab- 
bits, monkeys,  dogs,  and  cats  are  all  susceptible  to  experi- 
mental inoculation.  During  epidemics  the  purely  herbiv- 
orous animals  usually  escape,  though  oxen  have  been  known 
to  die  of  the  disease.  When  blood,  lymphatic  pulp,  or 
pure  cultures  are  inoculated  into  them,  the  animals  be- 
come ill  in  from  one  to  two  days,  according  to  their  size  and 
the  virulence  of  the  bacillus.  Their  eyes  become  watery, 
they  show  disinclination  to  take  food  or  to  make  any  bodily 
effort,  the  temperature  rises  to  41.5°  C.,  they  remain  quiet 
in  a  corner  of  the  cage,  and  die  with  convulsive  symptoms 
in  from  two  to  seven  days.  If  the  inoculation  be  made 
intravenously,  no  lymphatic  enlargement  occurs;  but  if  it 
be  made  subcutaneously,  the  nearest  lymph-nodes  always 
enlarge  and  suppurate  if  the  animal  live  long  enough.  The 
bacilli  are  found  everywhere  in  the  blood,  but  not  in  very 
large  numbers. 

Rats  seem  to  suffer  from  a  chronic  form  of  the  disease, 
and  sometimes  can  be  found  to  have  encapsulated  caseous 
nodules  in  the  submaxillary  glands,  caseous  bronchial  glands, 
and  fibroid  pneumonia,  months  after  inoculation.  In  all 
such  cases  virulent  plague  bacilli  are  present. 

In  and  about  San  Francisco  the  extermination  of  rats  for 
the  eradication  of  the  plague  was  unexpectedly  complicated 
by  the  discovery  that  other  rodents  with  which  the  rats  came 
into  contact  also  harbored  the  plague  bacilli.  McCoy  and 
Smith  f  found  this  to  be  true  of  the  prairie  dog,  the  desert 
wood  rat,  the  rock  squirrel,  and  the  brush  rat.  To  insure 
security  against  the  recurrence  of  the  disease  among  men 
necessitates  continued  observation  of  these  animals  and  the 
extermination  of  diseased  colonies,  as  well  as  their  complete 
extermination  in  the  neighborhood  of  human  habitations. 

According  to  Yersin,  an  infiltration  or  watery  edema  can 
be  observed  in  a  few  hours  about  the  point  of  inoculation. 
The  autopsy  shows  the  infiltration  to  be  made  up  of  a  yellow- 
ish gelatinous  exudation.  The  spleen  and  liver  are  enlarged, 
the  former  often  presenting  an  appearance  similar  to  that 

*  "Arbeiten  aus  d.  Kaiserl.  Gesundheitsamte,"  1901,  xvm. 
t  "Journal  of  Infectious  Diseases,"  1910,  vn,  p.  374. 


Experimental  Infection  591 

observed  in  miliary  tuberculosis.  Sometimes  there  is  uni- 
versal enlargement  of  the  lymphatic  glands.  Bacilli  are 
found  in  the  blood  and  in  all  the  internal  organs.  Skin 
eruptions  may  occur  during  life,  and  upon  the  inner  abdom- 
inal walls  petechiae  and  occasional  hemorrhages  may  be 
found.  The  intestine  is  hyperemic,  the  adrenals  congested. 
Serosanguinolent  effusions  may  occur  into  the  serous 
cavities. 

Devell*  has  found  frogs  susceptible  to  the  disease. 

Wyssokowitsch  and  Zabolotnyf  found  monkeys  highly 
susceptible  to  plague,  especially  when  subcutaneously  in- 
oculated. When  an  inoculation  was  made  with  a  pin  dipped 
in  a  culture  of  the  bacillus,  the  puncture  being  made  in  the 
palm  of  the  hand  or  sole  of  the  foot,  the  monkeys  always 
died  in  from  three  to  seven  days.  In  these  cases  the  local 
edema  observed  by  Yersin  did  not  occur.  They  point  out 
the  interest  attaching  to  infection  through  so  insignificant  a 
wound  and  without  local  lesions. 

Klein  {  found  that  intraperitoneal  injection  of  the  bacillus 
into  guinea-pigs  was  of  diagnostic  value,  producing  a  thick, 
cloudy,  peritoneal  exudate  rich  in  leukocytes  and  containing 
characteristic  chains  of  the  plague  bacillus,  occurring  in 
from  twenty-four  to  forty-eight  hours. 

The  plague  bacillus  may  enter  the  body  by  inhalation  from 
an  atmosphere  through  which  it  is  disseminated,  under 
which  circumstances  it  is  usually  of  the  pneumonic  type,  or 
it  may  enter  through  the  skin.  Lesions  too  small  to  be  seen 
with  the  naked  eye  may  suffice,  and  some  have  shown  that 
when  the  organisms  are  vigorously  rubbed  into  the  unbroken 
skin,  they  may  succeed  in  penetrating  it.  The  pulmonary 
or  pneumonic  form  is  not  unlike  other  forms  of  pneumonia. 
The  lung  is  consolidated,  enormous  numbers  of  plague  bacilli 
occur  in  the  sputum,  the  fever  is  high,  and  death  occurs  in  a 
few  days. 

Cases  in  which  the  infection  takes  place  through  the  skin 
soon  show  swelling  of  the  adjacent  lymph-nodes.  These 
become  very  large  and  tense,  occasion  great  suffering,  and 
finally  may  soften  and  evacuate  if  death  from  blood  invasion 
does  not  intervene. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Oct.  12,  1897. 
t  "Ann.  de  1'Inst.  Pasteur,"  Aug.  25,  1897,  xi,  8,  p.  665. 
}  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  xxi,  No.  24,  July  10,  1897, 
p.  849. 


592  Plague 

Mode  of  Infection. — Klein  found  that  animals  fed  upon 
cultures  of  the  bacillus  or  upon  the  flesh  of  animals  dead 
of  the  disease  became  ill  and  died  with  typical  symptoms. 
When  he  inoculated  animals  with  the  dust  of  dwelling-houses 
in  which  the  disease  had  occurred,  some  of  them  died  of 
tetanus,  one  from  plague.  Many  rats  and  mice  died  spon- 
taneously in  Hongkong,  examination  showing  the  character- 
istic bacilli.  Such  infected  animals  carry  the  cause  of  the 
disease  from  place  to  place  in  their  migrations. 

Yersin  showed  that  flies  may  die  of  the  disease.  Macer- 
ating and  crushing  a  fly  in  bouillon,  he  not  only  succeeded 
in  obtaining  the  bacillus,  but  infected  an  animal  with  it. 

Nuttall,*  in  repeating  Yersin 's  fly  experiment,  found  his 
observation  correct,  and  showed  that  flies  fed  with  the  ca- 
davers of  plague -infected  mice  die  in  a  variable  length  of  time. 
Large  numbers  of  plague  bacilli  were  found  in  their  intes- 
tines. He  also  found  that  bed-bugs  allowed  to  prey  upon 
infected  animals  took  up  large  numbers  of  the  plague  bacilli 
and  retained  them  for  a  number  of  days.  These  bugs  did 
not,  however,  infect  healthy  animals  when  allowed  to  bite 
them;  but  Nuttall  was  not  satisfied  that  the  number  of  his 
experiments  upon  this  point  was  great  enough  to  prove 
that  plague  cannot  be  spread  by  the  bites  of  suctorial 
insects. 

Ogata  found  plague  bacilli  in  fleas  taken  from  diseased 
rats.  He  crushed  some  fleas  between  sterile  object-glasses 
and  introduced  the  juice  into  the  subcutaneous  tissues  of 
a  mouse,  which  died  in  three  days  with  typical  plague,  a 
control-animal  remaining  well.  Some  guinea-pigs  taken  for 
experimental  purposes  into  a  plague  district  died  sponta- 
neously of  the  disease,  presumably  because  of  insect  in- 
fection. 

The  animal  most  prone  to  spontaneous  infection  seems  to 
be  the  rat,  and  there  is  much  evidence  in  support  of  the  view 
that  it  aids  in  the  spread  of  epidemics.  In  several  of  the 
Asiatic  plague  districts  and  at  Santos  the  appearance  of 
plague  among  the  inhabitants  was  preceded  by  a  large  mor- 
tality among  the  rats,  which  examination  showed  had  died 
of  plague.  Dead  rats  are  usually  to  be  found  in  plague- 
infected  houses  in  India. 

Galli- Valerio  f  and  others  think  that  the  fleas  of  the  mouse 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  xxi,  No.  24,  Aug.  13,  1897. 
t  Ibid.,  xxvii,  No.  i,  p.  i,  Jan.  6,  1900. 


Diagnosis  593 

and  rat  are  incapable  of  living  upon  man  and  do  not  bite 
him,  and  that  it  is  only  the  Pulex  irritans,  or  human  flea, 
that  can  transmit  the  disease  from  man  to  man.  Tidswell,* 
however,  found  that  of  100  fleas  collected  from  rats,  there 
were  four  species,  of  which  three — the  most  common  kinds 
— bit  men  as  well  as  rats.  Lisbon  f  found  that  of  246  fleas 
caught  on  men  in  the  absence  of  plague,  only  one  was  a  rat 
flea,  but  out  of  30  fleas  caught  upon  men  in  a  lodging-house, 
during  plague,  14  were  rat  fleas.  This  seems  to  show  that 
as  the  rats  die  off  their  fleas  seek  new  hosts,  and  may  thus 
contribute  to  the  spread  of  the  disease. 

That  fleas  can  cause  the  transmission  of  plague  from  animal 
to  animal  has  been  proved  by  experiments  made  in  India. 
These  experiments,  which  are  published  as  "Reports  on  Plague 
Investigations  in  India,"  issued  by  the  Advisory  Committee 
appointed  by  the  Secretary  of  State  for  India,  the  Royal 
Society,  and  the  Lister  Institute,  appear  in  the  Journal  of 
Hygiene  from  1 906  onward,  t  It  seems  from  these  experiments 
that  human  fleas  (Pulex  irritans)  do  not  bite  rats,  but  that 
the  rat  fleas  of  all  kinds  do,  though  not  willingly,  bite  men. 
By  placing  guinea-pigs  in  cages  upon  the  floor  of  infected 
houses,  the  fleas  of  all  kinds  quickly  attack  them  with  result- 
ing infection,  but  if  the  guinea-pigs  are  kept  in  flea-proof 
cages,  or  if  the  cages  are  surrounded  by  "tangle-foot,"  or 
"sticky  fly-paper,"  the  fleas,  not  being  able  to  spring  over  the 
barrier,  are  caught  on  the  sticky  surfaces  and  do  not  reach 
the  guinea-pigs,  which  then  remain  uninfected.  What  is 
true  of  the  guinea-pigs,  is  undoubtedly  true  of  the  rats;  the 
disease  is  transmitted  from  rat  to  rat  by  the  fleas  (Ceratophyl- 
lus  fasciatus,  Ctenopsylla  musculi,  and  Pulex  cheopis,  which 
appears  to  be  most  common). 

M.  Herzog§  has  shown  that  pediculi  may  harbor  plague 
bacilli  and  act  as  carriers  of  the  disease. 

The  plague  bacilli  can  also  be  transmitted  from  place  to 
place  by  fomites,  from  which  they  may  reach  man  or  rats. 

Diagnosis. — It  seems  possible  to  make  a  diagnosis  of  the 
disease  in  doubtful  cases  by  examining  the  blood,  but  it  is 

*" British  Medical  Journal,"  June  27,  1903. 

f  "Times  of  India,"  Nov.  26,  1904. 

t  "Journal  of  Hygiene,"  Sept.,  1906,  vol.  vi,  p.  421;  July,  1907,  vol. 
vn,  p.  324;  Dec.,  1907,  vol.  vn,  p.  693;  May,  1908,  vol.  vin,  p.  162; 
1909,  vol.  ix;  1910,  vol.  x;  1911,  vol.  xi. 

§  "Amer.  Jour.  Med.  Sci.,"  March,  1895. 
38 


594  Plague 

admitted  that  a  good  deal  of  bacteriologic  practice  is  neces- 
sary for  the  purpose. 

Abel  found  that  blood-examinations  may  yield  doubt- 
ful results  because  of  the  variable  appearance  of  the  con- 
tained bacilli,  which  may  easily  be  mistaken  for  other  bac- 
teria. He  deems  the  best  tests  to  be  the  inoculation  of 
broth  cultures  and  the  subsequent  inoculation  into  animals, 
which,  he  advises,  should  have  been  previously  vaccinated 
against  the  streptococcus. 

Kolle*  has  suggested  a  method  valuable  both  for  the 
diagnosis  of  the  disease  and  for  estimating  the  virulence  of 
the  bacillus.  It  is  as  follows:  "The  skin  over  a  portion  of 
the  abdominal  wall  of  the  guinea-pig  is  shaved,  care  being 
taken  to  avoid  the  slightest  injury  of  the  skin.  The  infective 
material  is  carefully  rubbed  into  the  shaved  skin.  Im- 
portant, in  order  rightly  to  understand  the  occurrence  of 
plague  infection,  is  the  fact  disclosed  here  in  the  case  of 
guinea-pigs,  that  by  this  method  of  inoculation  the  animals 
present  the  picture  of  true  bubonic  plague — that  is  to  say, 
the  production  of  nodules  in  the  various  organs,  principally 
in  the  spleen.  In  this  manner  guinea-pigs,  which  would 
not  be  affected  by  large  subcutaneous  injections,  even 
amounting  to  2  mg.  of  agar  culture  (equal  to  a  loop)  of  low- 
virulence  plague  bacillus,  may  be  infected  and  eventually 
succumb." 

Virulence. — By  frequent  passage  through  animals  of  the 
same  species  the  bacillus  can  be  much  increased  in  virulence. 
Kolle  recommends  rats  for  this  purpose,  and,  indeed,  declares 
that  without  the  use  of  rats  it  is  impossible  to  keep  cultures 
at  a  high  grade  of  virulence.  Batzaroff  found  that  the  most 
virulent  plague  bacilli  were  to  be  obtained  from  the  pneu- 
monic lungs  of  rats  that  had  been  infected  through  the  nasal 
aperture  with  cotton-wool  saturated  with  a  culture  of  the 
bacillus.  This  is  not,  however,  a  reliable  method  of  inocu- 
lation. 

Yersin  found  that  when  cultivated  for  any  length  of  time 
upon  culture-media,  especially  agar-agar,  the  virulence  was 
rapidly  lost  and  the  bacillus  eventually  died.  On  the  other 
hand,  when  constantly  inoculated  from  animal  to  animal, 
the  virulence  of  the  bacillus  is  much  increased. 

*  See  Havelburg,  "Public  Health  Reports,"  Aug.  15,  1902,  vol. 
xvn,  No.  33,  p.  1863. 


Sanitation  595 

Knorr,  Yersin,  Calmette,  and  Borrel*  have  shown  that 
the  bacillus  made  virulent  by  frequent  passage  through  mice 
is  not  increased  in  virulence  for  rabbits. 

This  no  doubt  depends  upon  the  sensitivity  of  the  bacillus 
to  the  protective  substances  of  the  body  juices,  immuniza- 
tion against  those  of  one  animal  not  necessarily  protecting 
the  organism  against  those  of  other  animals. 

Bielonovskyf  finds  that  broth,  agar,  and  serum  cultures 
of  the  plague  bacillus  possess  the  property  of  hemolyzing 
the  blood  of  normal  animals.  The  hemolytic  power  of  fil- 
trates of  plague  cultures  increases  up  to  the  thirteenth  or 
fourteenth  day,  then  gradually  diminishes,  but  without 
completely  disappearing.  The  hemolysins  are  notably  re- 
sistant to  heat,  not  being  destroyed  below  100°  C. 

Sanitation. — A  disease  that  may  be  transmitted  from 
man  to  man  by  atmospheric  infection  and  inhalation,  that  can 
be  transported  from  place  to  place  by  fomites,  that  occurs  in 
epidemic  form  among  the  lower  animals  as  well  as  among  men, 
and  that  can  be  transmitted  from  man  to  man  and  from  lower 
animals  to  man  by  biting  insects,  must  inevitably  become  a 
source  of  anxiety  to  the  sanitarian. 

The  preventive  measures  must  take  account  of  men,  rats, 
and  goods.  If  vessels  are  permitted  to  visit  and  leave  plague- 
stricken  ports,  means  must  be  taken  to  see  that  all  passengers 
are  healthy  at  the  time  of  leaving  and  have  remained  so 
during  the  voyage,  and  provision  should  be  made  at  the  port 
of  entry  for  the  disinfection  of  the  cargo  before  the  foods  are 
landed.  But  the  rats  must  be  given  special  consideration, 
for  so  soon  as  the  vessel  reaches  port  some  of  them  jump  over- 
board and  swim  to  the  shore,  carrying  the  disease  with  them. 
When  a  vessel  visits  a  plague  port,  every  precaution  should  be 
taken  to  orevent  the  entrance  of  rats,  first  by  anchoring 
in  the  stream  instead  of  tying  to  the  dock ;  by  carefully  scru- 
tinizing the  packages  taken  from  the  lighters  to  see  that  there 
are  no  rats  hidden  among  them ;  by  placing  large  metal  shields 
or  reversed  funnels  about  all  anchor  chains,  hawsers,  and 
cables  so  that  no  rats  can  climb  up  from  the  water  in  which 
they  are  swimming  at  night.  Arrangements  should  also  be 
made  for  rat  destruction  on  board  the  ship  by  means  of  sul- 
phurous oxid  or  other  poisonous  vapors  to  rid  the  ship  of 
rats  before  the  next  port  is  reached.  Passengers  and  crew 

*  "Ann.  de  1'Inst.  Pasteur.,"  July,  1895. 

t  "Arch,  des  Sci.  Biol.,"  Tome  x,  No.  4,  St.  Petersb.,  1904. 


596  Plague 

should  also  be  kept  in  quarantine  before  mingling  with 
society.  It  is  much  more  easy  to  keep  plague  out  of  a 
port  than  to  combat  it  when  it  has  entered,  for  under  the 
latter  condition  are  involved  the  isolation  of  the  patients 
in  rat-free  and  vermin-free  quarters,  the  disinfection  of 
the  premises  and  goods  where  the  case  arose,  and  an  im- 
mediate warfare  upon  the  rats  and  other  small  animals  of  the 
neighborhood.  To  emphasize  how  difficult  the  latter  may  be 
it  is  only  necessary  to  point  out  that  plague  reached  San 
Francisco  in  May,  1907,  during  which  year  there  were  156 
cases  and  76  deaths.  Every  precaution  was  taken  to  prevent 
its  spread,  and  extermination  of  rats,  many  being  found 
infected,  practised.  Though  at  great  expense  and  with  the 
utmost  thoroughness  the  rats  were  destroyed,  the  plague 
spread  to  the  ground  squirrels  and  other  small  rodents,  and  in 
1911  plague-infected  rodents  were  still  to  be  found  in  the 
outskirts  of  the  city. 

Immunity. — An  attack  of  plague  usually  exempts  from 
future  attacks.  Kitasato's  experiments  first  showed  that  it 
was  possible  to  bring  about  immunity  against  the  disease,  and 
Yersin,  working  in  India,  and  Fitzpatrick,  in  New  York, 
have  successfully  immunized  large  animals  (horses,  sheep, 
and  goats).  The  serum  of  the  immunized  animals  contains 
specific  agglutinins  and  bacteriolysins  as  well  as  an  antitoxin, 
capable  not  only  of  preventing  the  disease,  but  also  of  curing 
it  in  mice  and  guinea-pigs  and  probably  in  man. 

Haffkine's  Prophylactic. — Haffkine*  followed  his  plan 
of  preventive  inoculation  as  employed  against  cholera,  and 
has  invented  a  mode  of  prophylaxis  based  upon  the  use 
of  devitalized  cultures.  Bouillon  cultures  are  used,  and 
small  floating  drops  of  butter  are  employed  to  make  the 
"islands"  of  plague  bacilli  float.  The  cultures  are  grown 
for  a  month  or  so,  successive  crops  of  the  island-stalactite 
growth  being  precipitated  by  agitating  the  tube.  In  this 
manner  an  "intense  extracellular  toxin"  containing  large 
numbers  of  the  bacilli  is  prepared.  The  culture  was  killed 
by  exposure  to  70°  C.  for  one  hour,  and  used  in  doses  of  i  to  3 
c.c.  as  a  preventive  inoculation. 

An  interesting  collection  of  statistics,  showing  in  a  con- 
vincing manner  the  value  of  the  Haffkine  prophylactic,  is 
published  of  Leumann,  of  Hubli.  The  figures,  together 

*  "Brit.  Med.  Jour.,"  June  12,  1897. 


Haffkine  Prophylactic  597 

with  a  great  deal  of  interesting  information  upon  the  subject, 
can  be  found  in  the  paper  upon  "A  Visit  to  the  Plague  Dis- 
tricts in  India,"  by  Barker  and  Flint.* 

The  immunity  conferred  by  the  Haffkine  prophylactic 
lasts  about  a  month.  The  preparation  must  never  be  used 
if  the  person  has  already  been  exposed  to  infection,  and  is 
in  the  incubation  stage  of  the  disease,  as  it  contains  the 
toxins  of  the  disease,  and  therefore  greatly  intensifies  the 
existing  condition.  When  injected  into  healthy  persons 
it  always  produces  some  fever,  slight  local  swellings,  and 
malaise. 

Wyssokowitsch  and  Zabolotny,f  whose  studies  have  al- 
ready been  quoted,  used  96  monkeys  in  the  study  of  the  value 
of  the  "plague  serums,"  and  found  that  when  treatment  is 
begun  within  two  days  from  the  time  of  inoculation  the 
animals  can  be  saved,  even  though  symptoms  of  the  disease 
are  marked.  After  the  second  day  the  treatment  cannot 
be  relied  upon.  The  dose  necessary  was  20  c.c.  of  a  serum 
having  a  potency  of  i  :  10.  If  too  little  serum  was  given, 
the  course  of  the  disease  was  retarded  and  the  animal  im- 
proved for  a  time,  then  suffered  a  relapse,  and  died  in  from 
thirteen  to  seventeen  days.  The  serum  also  produced 
immunity,  but  of  only  ten  to  fourteen  days'  duration.  Im- 
munity lasting  three  weeks  was  conferred  by  inoculating 
a  monkey  with  an  agar-agar  culture  heated  to  60°  C.  If 
too  large  a  dose  of  such  a  culture  was  given,  however,  the 
animal  was  enfeebled  and  remained  susceptible. 

Of  Yersin's  serum,  which  is  prepared  by  immunizing 
horses  against  the  toxins  and  cultures  of  the  bacillus  in  the 
usual  manner,  5  c.c.  doses  have  been  found  to  confer  an  im- 
munity lasting  for  about  a  fortnight.  Larger  doses  confer  a 
longer  immunity.  For  the  treatment  of  the  developed 
disease  in  man,  doses  of  50  and  even  100  to  200  c.c.  seem 
necessary  to  produce  the  desired  effect. 

OTHER  MICRO-ORGANISMS  OF  THE  PLAGUE  GROUP. 

The  Bacillus  pestis  is  a  member  of  a  group  of  organisms 
collectively  known  as  the  bacilli  of  hemorrhagic  septicemia. 
Two  of  these  organisms  are  of  sufficient  interest  to  deserve 
special  mention. 

*  "New  York  Med.  Jour.,"  Feb.  3,  1900. 
t  Loc.  cit. 


598        Micro-organisms  of  the  Plague  Group 

BACILLUS  CHOLERA  GALLINARUM  (PERRONCITO)  ;  BACILLUS 

CHOLERA;   BACILLUS  AVICIDUM;   BACILLUS  Avi- 

SEPTICUS;  BACILLUS  OF  RABBIT  SEPTI- 

CEMIA;  BACILLUS  CUNICULICIDA. 

General  Characteristics.— A  non-motile,  non-flagellated,  non- 
sporogenous,  non-liquefying,  non-chromogenic,  aerobic  bacillus,  patho- 
genic for  birds  and  mammals,  staining  by  the  ordinary  methods,  but 
not  by  Gram's  method,  producing  acids,  indol,  and  phenol,  and  co- 
agulating milk. 

The  barnyards  of  both  Europe  and  America  are  occa- 
sionally visited  by  an  epidemic  disease  known  as  "  chicken- 
cholera,"  Huhner  cholera,  or  cholera  de  poule,  which  rapidly  de- 
stroys pigeons,  turkeys,  chickens,  ducks,  and  geese.  Rabbit- 
warrens  are  also  at  times  affected  and  the  rabbits  killed. 

The  bacillus  responsible  for  this  disease  was  first  observed 
by  Perroncito*  in  1878,  and  afterward  thoroughly  studied 
by  Toussaint  and  Pasteur,  f 

Morphology. — The  organisms  are  short  and  broad,  with 
rounded  ends,  measuring  i  X  0.4  to  0.6  ^,  sometimes  joined 
to  produce  chains.  Pasteur  at  first  Regarded  them  as  diplo- 
cocci,  because  the  poles  stain  intensely,  a  narrow  space 
between  them  remaining  almost  uncolored.  This  pecu- 
liarity is  very  marked,  and  careful  examination  is  required 
to  detect  the  intermediate  substance.  The  bacillus  does  not 
form  spores,  is  not  motile,  and  has  no  flagella.  | 

Staining. — The  organism  stains  with  ordinary  anilin  dye 
solutions,  but  not  by  Gram's  method. 

Cultivation. — Colonies. — Colonies  upon  gelatin  plates 
appear  after  about  two  days  as  small,  irregular,  white  points. 
The  deep  colonies  reach  the  surface  slowly,  and  do  not  attain 
to  any  considerable  size.  The  gelatin  is  not  liquefied.  The 
colonies  appear  under  the  microscope  as  irregularly  rounded 
yellowish-brown  disks  with  distinct  smooth  borders  and 
granular  contents.  Sometimes  there  is  a  distinct  concentric 
arrangement. 

Gelatin. — In  gelatin  puncture  cultures  a  delicate  white 
line  occurs  along  the  entire  path  of  the  wire.  Upon  the 
surface  the  development  is  much  more  marked,  so  that  the 
growth  resembles  a  nail  with  a  good-sized  flat  head.  If 

*  "Archiv.  f.  wissenschaftliche  und  praktische  Thierheilkunde,"  1879. 
t  "  Compte-rendu  de  1'Acad.  de  Sci.  de  Paris,"  vol.  xc. 
J  Thoinot  and  Masselin  assert  that  the  organism  is  motile.     "Precis 
de  Microbie,"  2d  ed.,  1893. 


Chicken  Cholera  599 

the  bacilli  be  planted  upon  the  surface  of  obliquely  solidi- 
fied gelatin,  a  much  more  pronounced  growth  takes  place, 
and  along  the  line  of  inoculation  a  dry,  granular  coating  is 
formed.  There  is  no  liquefation  of  the  medium. 

Bouillon. — The  growth  in  bouillon  is  accompanied  by  a 
slight  cloudiness. 

Agar. — This  growth,  like  that  upon  agar-agar  and  blood- 


Fig.   207. — Bacillus  of  chicken-cholera,   from  the  heart's  blood  of  a 
pigeon.    X    1000    (Frankel  and   Pfeiffer). 

serum,  is  white,  shining,  rather  luxuriant,  and  devoid  of 
characteristics . 

Potato. — Upon  potato  no  growth  occurs  except  at  37°  C. 
It  is  a  very  insignificant,  yellowish-gray,  translucent  film. 

Milk  is  acidulated  and  slowly  coagulated. 

Vital  Resistance. — The  bacillus  readily  succumbs  to  the 
action  of  heat  and  dry  ness.  The  organism  is  an  obligatory 
ae'robe. 

Metabolic  Products. — Indol  and  phenol  are  formed, 
Acids  are  produced  in  sugar-containing  media,  without  gas 
formation. 


6oo       Micro-organisms  of  the  Plague  Group 

Pathogenesis. — The  introduction  of  cultures  of  this 
bacillus  into  chickens,  geese,  pigeons,  sparrows,  mice,  and 
rabbits  is  sufficient  to  produce  fatal  septicemia.  Feeding 
chickens,  pigeons,  and  rabbits  with  material  infected  with 
the  bacillus  is  also  sufficient  to  produce  the  disease.  Guinea- 
pigs,  cats,  and  dogs  seem  immune,  though  they  may  succumb 
to  large  doses  if  given  intraperitoneally.  The  organism  is 
probably  harmless  to  man. 

Fowls  ill  with  the  disease  fall  into  a  condition  of  weakness 
and  apathy,  which  causes  them  to  remain  quiet,  seemingly 
almost  paralyzed,  and  the  feathers  ruffled  up.  The  eyes 
are  closed  shortly  after  the  illness  begins,  and  the  birds 
gradually  fall  into  a  stupor,  from  which  they  do  not  awaken. 
The  disease  is  fatal  in  from  twenty-four  to  forty -eight  hours. 
During  its  course  there  is  profuse  diarrhea,  with  very  fre- 
quent fluid,  slimy,  grayish-white  discharges. 

Lesions. — The  autopsy  shows  that  when  the  bacilli  are 
introduced  subcutaneously  a  true  septicemia  results,  with 
the  formation  of  a  hemorrhagic  exudate  and  gelatinous 
infiltration  at  the  seat  of  inoculation.  The  liver  and  spleen 
are  enlarged;  circumscribed,  hemorrhagic,  and  infiltrated 
areas  occur  in  the  lungs;  the  intestines  show  an  intense 
inflammation  with  red  and  swollen  mucosa,  and  occasional 
ulcers  following  small  hemorrhages.  Pericarditis  is  fre- 
quent. The  bacilli  are  found  in  all  the  organs.  If,  on  the 
other  hand,  the  disease  has  been  produced  by  feeding,  the 
bacilli  are  chiefly  to  be  found  in  the  intestine.  Pasteur 
found  that  when  pigeons  were  inoculated  into  the  pectoral 
muscles,  if  death  did  not  come  on  rapidly,  portions  of  the 
muscle  (sequestra)  underwent  degeneration  and  appeared 
anemic,  indurated,  and  of  a  yellowish  color. 

Immunity. — Pasteur*  discovered  that  when  cultures  are 
allowed  to  remain  undisturbed  for  several  months,  their 
virulence  becomes  greatly  lessened,  and  new  cultures  trans- 
planted from  them  are  also  attenuated.  If  chickens  be 
inoculated  with  such  attenuated  cultures,  no  other  change 
occurs  than  a  local  inflammatory  reaction  that  soon  disap- 
pears and  leaves  the  birds  protected  against  future  infec- 
tion with  virulent  bacilli.  From  these  observations  Pasteur 

*  An  interesting  account  of  Pasteur's  experiments  upon  chicken-chol- 
era can  be  found  in  the  "Life  of  Pasteur, "  by  Vallery-Radot,  translated 
by  Mrs.  R.  S.  Devonshire,  1909.  Popular  Edition,  New  York,  Double- 
day,  Page  and  Co. 


Swine-plague  60 1 

worked  out  a  system  of  protective  vaccination  in  which  the 
fowls  are  first  inoculated  with  attenuated,  then  with  more 
active,  and  finally  with  virulent,  cultures,  with  resulting 
protection  and  immunity. 

Use  has  been  made  of  this  bacillus  to  kill  rabbits  in  Aus- 
tralia, where  they  are  pests.  It  is  estimated  that  two  gallons 
of  bouillon  culture  will  destroy  20,000  rabbits,  irrespective 
of  infection  by  contagion. 

The  bacillus  of  chicken-cholera  may  be  identical  with 
organisms  found  in  various  epidemic  diseases  of  larger 
animals,  and,  indeed,  no  little  confusion  has  arisen  from 
the  description  of  what  is  now  pretty  generally  accepted  to 
be  the  same  organism  as  the  bacillus  of  rabbit  septicemia 
(Koch),  Bacillus  cuniculicida  (Fliigge),  bacillus  of  swine- 
plague  (Loffler  and  Schiitz),  bacillus  of  "Wildseuche" 
(Hiippe),  bacillus  of  "Biiffelseuche"  (Oriste-Armanni),  etc. 


BACILLUS  SUISEPTICUS  (LOFFLER  AND  SCHUTZ). 

General  Characteristics. — A  non-motile,  non-flagellated,  non- 
sporogenous,  non-liquefying,  non-chromogenic,  aerobic  and  optionally 
anae'robic  bacillus,  pathogenic  for  hogs  and  many  other  animals,  stain- 
ing by  the  ordinary  methods,  but  not  by  Gram's  method.  It  produces 
a  slight  acidity  in  milk,  but  does  not  coagulate  it. 

The  bacillus  of  swine-plague,  or  Bacillus  suisepticus  of 
Loffler  and  Schiitz*  and  Salmon  and  Smith,  f  but  slightly 
resembles  the  bacillus  of  hog-cholera  (q.  v.),  though  it  was 
formerly  confounded  with  it  and  at  one  time  thought  to  be 
identical  with  it.  The  species  have  sufficient  well-marked 
characteristics,  however,  to  make  their  differentiation  easy. 

Swine-plague  is  a  rather  common  and  exceedingly  fatal 
epidemic  disease.  It  not  infrequently  occurs  in  association 
with  hog-cholera,  and  because  of  the  lack  of  sufficiently  well- 
characterized  symptoms — sick  hogs  appearing  more  or  less 
alike — is  often  mistaken  for  it.  The  confusion  resulting  from 
such  faulty  diagnosis  makes  it  difficult  to  determine  exactly 
how  fatal  either  may  be  in  uncomplicated  cases. 

Morphology. — The  bacillus  of  swine-plague  much  re- 
sembles that  of  hog-cholera,  and  not  a  little  that  of  chicken- 
cholera.  It  is  a  short  organism,  rather  more  slender  than 

:  "Arbeiten  aus  den  kaiserlichen  Gesundheitsamte,"  I. 
t  "Zeitschrift  f.  Hygiene,"  x. 


602        Micro-organisms  of  the  Plague  Group 

the  related  species,  not  possessed  of  flagella,  incapable  of 
movement,  and  producing  no  spores. 

It  is  an  optional  anaerobe. 

Staining. — The  bacillus  stains  by  the  ordinary  methods, 
sometimes  only  at  the  poles,  then  closely  resembling  the 
bacillus  of  chicken-cholera.  It  is  not  colored  by  Gram's 
method. 

Cultivation. — In  general,  the  appearance  in  culture-media 
is  very  similar  to  that  of  the  hog  cholera  bacillus.  Kruse,* 
however,  points  out  that  when  the  bacillus  grows  in  bouillon 
the  liquid  remains  clear,  the  bacteria  gathering  to  form  a 
flocculent,  stringy  sediment.  The  organism  does  not  grow 
upon  ordinary  acid  potato,  but  if  the  reaction  of  the  me- 
dium be  alkaline,  a  grayish-yellow  patch  is  formed.  In  milk 
a  slight  acidity  is  produced,  but  the  milk  is  not  coagu- 
lated. 

Vital  Resistance. — The  vitality  of  the  organism  is  low, 
and  it  is  easily  destroyed.  Salmon  says  that  it  soon  dies  in 
water  or  when  dried,  and  that  the  temperature  for  its  growth 
must  be  more  constant  and  every  condition  of  life  more 
favorable  than  for  the  hog-cholera  germ.  The  organism  is 
said  to  be  widely  distributed  in  nature,  and  is  probably 
present  in  every  herd  of  swine,  though  not  pathogenic  except 
when  its  virulence  becomes  increased  or  the  vital  resistance 
of  the  animals  diminished  by  some  unusual  condition. 

Pathogenesis. — While  similar  to  hog-cholera,  swine- 
plague  presents  some  marked  differences,  especially  in 
regard  to  the  seat  of  the  local  manifestations,  and  in  its 
duration,  which  is  much  shorter.  There  is  also  considerable 
resemblance  to  chicken-cholera,  but  the  local  reaction  fol- 
lowing the  injection  of  the  micro-organisms  partakes  of  the 
nature  of  a  hemorrhagic  edema,  which  is  not  present  in 
chicken-cholera,  and  rabbits  commonly  exhibit  fatty  meta- 
morphosis of  the  liver. 

Rabbits,  mice,  and  small  birds  are  very  susceptible  to 
the  disease,  usually  dying  of  septicemia  in  twenty-four 
hours;  guinea-pigs  are  less  susceptible,  except  very  young 
animals,  which  die  without  exception.  Chickens  are  more 
immune,  but  usually  succumb  to  large  doses.  Hogs  die  of 
septicemia  after  subcutaneous  injection  of  the  bacilli.  There 
is  a  marked  edema  at  the  point  of  injection.  If  injected 
into  the  lung,  a  pleuropneumonia  follows,  with  multiple 
*  Fliigge's  "Die  Mikroorganismen,"  p.  419,  1896. 


Swine-plague  603 

necrotic  areas  in  the  lung.  In  these  cases  the  spleen  is  not 
much  swollen,  there  is  slight  gastro-intestinal  catarrh,  and 
the  bacilli  are  present  everywhere  in  the  blood. 

Animals  can  be  infected  only  by  subcutaneous,  intra- 
venous, and  intraperitoneal  inoculation,  not  by  feeding. 

As  seen  in  hogs,  the  symptoms  of  swine-plague  closely 
resemble  those  of  hog-cholera,  but  differ  in  the  existence  of 
cough,  swine-plague  being  prone  to  affect  the  lungs  and 
oppress  the  breathing,  which  becomes  frequent,  labored, 
and  painful,  while  hog-cholera  is  chiefly  characterized  by 
intestinal  symptoms. 

The  course  of  the  disease  is  usually  rapid,  and  it  may  be 
fatal  in  a  day  or  two. 

Lesions. — At  autopsy  the  lungs  are  found  to  be  inflamed, 
and  to  contain  numerous  small,  pale,  necrotic  areas,  -and 
sometimes  large  cheesy  masses  one  or  two  inches  in  diameter. 
Inflammations  of  the  serous  membranes  affecting  the  pleura, 
pericardium,  and  peritoneum,  and  associated  with  fibrinous 
inflammatory  deposits  on  the  surfaces,  are  common.  There 
may  be  congestion  of  the  mucous  membrane  of  the  intes- 
tines, particularly  of  the  large  intestine,  or  the  disease  in 
this  region  may  be  an  intense  croupous  inflammation  with 
the  formation  of  a  fibrinous  exudative  deposit  on  the  surface. 
A  hemorrhagic  form  of  the  disease  is  said  to  be  common 
in  Europe,  but,  according  to  Salmon,  is  rare  in  the  United 
States. 


CHAPTER   XXIV. 
ASIATIC  CHOLERA. 

SPIRILLUM  CHOLERA  ASIATICS  (Kocn*). 

General  Characteristics. — A  motile,  flagellated,  non-sporogenoust 
liquefying,  non-chromogenic,  non-aerogenic,  parasitic  and  saprophytic, 
pathogenic,  aerobic  and  optionally  anaerobic  spirillum,  staining  by 
ordinary  methods,  but  not  by  Gram's  method. 

Cholera  is  a  disease  endemic  in  certain  parts  of  India 
and  probably  indigenous  in  that  country.  Though  early 
mention  of  it  was  made  in  the  letters  of  travelers,  and  though 
it  appeared  in  medical  literature  and  in  governmental 
statistics  more  than  a  century  ago,  we  find  that  little 
attention  was  paid  to  the  disease,  except  in  its  disastrous 
effect  upon  the  armies,  native  and  European,  of  India  and 
adjacent  countries.  The  opening  up  of  India  by  Great 
Britain  in  the  last  half  century  made  scientific  observation 
of  the  disease  possible  and  permitted  us  to  determine  the 
relation  its  epidemics  bear  to  the  manners  and  customs 
of  the  people. 

The  filthy  habits  of  the  Oriental  people,  their  poverty, 
crowded  condition,  and  peculiar  religious  customs,  are  all 
found  to  aid  in  the  distribution  of  the  disease.  Thus,  the 
city  of  Benares  drains  into  the  Ganges  River  by  a  mcst 
imperfect  system,  which  distributes  the  greater  part  of 
the  sewage  immediately  below  the  banks  upon  which  the 
city  is  built  and  along  which  are  the  numerous  "Ghats" 
or  staircases  by  which  the  people  reach  the  sacred  waters. 
It  is  a  matter  of  religious  observance  for  every  zealot  who 
makes  a  pilgrimage  to  the  "  sacred  city"  to  take  a  bath  in 
and  drink  a  quantity  of  this  sacred  but  polluted  water, 
and  it  may  be  imagined  that  the  number  of  pious  Hindoos 
who  leave  Benares  with  "comma  bacilli"  in  their  intestines 
or  upon  their  clothes  must  be  great,  for  there  are  few  months 
in  the  year  when  the  city  is  exempt  from  cholera. 

*  "Deutsche  med.  Wochenschrift,"  1884-1885,  Nos.  19,  20,  37,  38, 
and  39. 

604 


Distribution  605 

The  pilgrimages  and  great  festivals  of  the  Hindoos  and 
Moslems,  by  bringing  together  enormous  numbers  of  people 
to  crowd  in  close  quarters  where  filth  and  bad  diet  prevail, 
cause  a  rapid  increase  in  the  number  of  cases  during  these 
periods  and  facilitate  the  distribution  of  the  disease  when 
the  festivals  break  up.  Probably  no  more  favorable  con- 
ditions for  the  dissemination  of  a  disease  can  be  imagined 
than  occurs  with  the  return  of  the  Moslem  pilgrims  from 
Mecca.  The  disease  extends  readily  along  the  regular  lines 
of  travel,  visiting  town  after  town,  until  from  Asia  it  has 
frequently  extended  into  Europe,  and  by  steamships  plying 
foreign  waters  has  several  times  been  carried  to  our  own 
continent.  Many  cases  are  on  record  which  show  conclu- 
sively how  a  single  ship,  having  a  few  cholera  cases  on 
board,  may  be  the  starting-point  of  an  outbreak  of  the 
disease  in  the  port  at  which  it  arrives. 

The  most  recent  great  epidemic  of  cholera  began  in  1883. 
From  Asia  it  spread  westward  throughout  Europe,  extended 
by  means  of  the  steamship  lines  to  numerous  of  the  large 
ports,  of  which  Hamburg  in  Germany  suffered  most  acutely, 
and  even  extended  to  some  of  the  ports  of  Africa  and  America. 
Russia  probably  suffered  more  than  any  other  European 
country,  and  it  is  estimated  that  in  that  country  there  were 
no  less  than  800,000  deaths.  During  1911  the  disease 
again  appeared  in  Europe  and  invaded  the  countries  along 
the  Mediterranean  coasts. 

Specific  Organism. — The  discovery  of  the  spirillum  of 
cholera  was  made  by  Koch  while  serving  as  a  member  of  a 
German  commission  appointed  to  study  the  disease  in  Egypt 
and  India  in  1883-84.  Since  its  discovery  the  spirillum  has 
been  subjected  to  much  careful  investigation,  and  an  im- 
mense amount  of  literature,  a  large  part  of  which  was  stim- 
ulated by  the  Hamburg  epidemic  of  1892,  has  accumulated. 

Distribution. — The  cholera  spirilla  can  be  found  with 
great  regularity  in  the  intestinal  evacuations  of  cholera 
cases,  and  can  often  be  found  in  drinking-water  and  milk, 
and  upon  vegetables,  etc.,  in  cholera-infected  districts. 
There  can  be  little  doubt  that  they  find  their  way  into 
the  body  with  the  food  and  drink.  Cases  in  the  literature 
show  how  cholera  germs  enter  drinking-water  and  are  thus 
distributed ;  how  they  are  sometimes  thoughtlessly  sprinkled 
over  green  vegetables  offered  for  sale  in  the  streets,  with 
infected  water  from  polluted  gutters;  how  they  enter  milk 
with  water  used  to  dilute  it ;  how  they  appear  to  be  carried 


6o6 


Asiatic  Cholera 


about  in  clothing  and  upon  food-stuffs;  how  they  can  be 
brought  to  articles  of  food  by  flies  that  have  preyed  upon 
cholera  excrement ;  and  other  interesting  modes  of  infection. 
The  literature  is  so  vast  that  it  is  scarcely  possible  to  men- 
tion even  the  most  instructive  examples.  A  bacteriologist 
became  infected  while  experimenting  with  the  cholera  spirilla 
in  Koch's  laboratory.  It  is  commonly  supposed  that  the 
cholera  organism  may  remain  alive  in  water  for  an  almost 
unlimited  length  of  time,  but  experiments  have  not  shown 
this  to  be  the  case.  Thus,  Wolffhiigel  and  Riedel  have 
shown  that  if  the  spirilla  be  planted  in  sterilized  water  they 
grow  with  great  rapidity  after  a  short  time,  and  can  be  found 
alive  after  months  have  passed.  Frankel,  however,  points 
out  that  this  ability  to  grow  and  remain  vital  for  long  peri- 
ods in  sterilized  water  does  not  guarantee  the  same  power 
of  growth  in  unsterilized  water,  for  in  the  latter  the  simul- 
taneous growth  of  other  bacteria  serves  to  extinguish  the 
cholera  spirilla  in  a  few  days. 


J 

J 


Fig.  208. — Cholera  spirilla. 

Morphology. — The  micro-organism  described  by  Koch, 
and  now  generally  accepted  to  be  the  cause  of  cholera,  is 
a  short  rod  i  to  2  ^  in  length  and  0.5  ^  in  breadth,  with  rounded 
ends,  and  a  distinct  curve,  so  that  the  original  name  by  which 
it  was  known,  the  "  comma  bacillus,"  applies  very  well.  One 
of  the  most  common  forms  is  that  in  which  two  short  curved 
individuals  are  conjoined  in  an  S-shaped  curve. 

When   the   conditions   of   nutrition    are   good,    multipli- 


The  Comma  Bacillus  607 

cation  by  fission  progresses  with  rapidity;  but  when  ad- 
verse conditions  arise,  long  spiral  threads — unmistak- 
able spirilla — develop.  Frankel  found  that  the  exposure 
of  the  cultures  to  unusually  high  temperatures,  the  addition 
of  small  amounts  of  alcohol  to  the  culture-media,  and  other 
unfavorable  conditions  lead  to  the  production  of  spirals 
instead  of  "commas." 

The  cholera  spirilla  are  actively  motile,  and  in  hanging- 
drop  preparations  can  be  seen  to  swim  about  with  great 
rapidity.  Both  comma- shaped  and  spiral  organisms  move 
with  a  rapid  rotary  motion. 

The  presence  of  flagella  can  be  demonstrated  without 


*£•&**** 

#rk '.. 


Coa*«         <i.t\ 

s*-*  ;%%• 


Fig.  209. — Spirillum  of  Asiatic  cholera,  from  a  bouillon  culture  three 
weeks  old,  showing  long  spirals.     X  1000  (Frankel  and  Pfeiffer). 


difficulty.  Each  spirillum  possesses  a  single  nagellum 
attached  to  one  end  (spiromonas). 

Involution-forms  of  bizarre  appearance  are  common  in 
old  cultures  of  the  spirillum,  and  sometimes  in  fresh  cultures 
many  individuals  show  by  granular  cytoplasm  and  irregular 
outline  that  they  are  degenerated.  Cholera  spirilla  from 
various  sources  differ  in  the  extent  of  involution. 

In  partially  degenerated  cultures  containing  long  spirals 
Hiippe  observed,  by  examination  in  the  "hanging-drop," 
certain  large  spheric  bodies  which  he  described  as  spores 
(arthrospores).  Koch  and,  indeed,  all  other  observers  fail 


608 


Asiatic  Cholera 


to  find  spores  in  the  cholera  organism,  and  the  nature  of 
the  bodies  described  by  Hiippe  must  be  regarded  as  doubtful. 
Staining.  —  The  cholera  spirillum  stains  well  with  the 
ordinary  aqueous  solutions  of  the  anilin  dyes,  especially 
fuchsin.  At  times  the  staining  must  be  continued  for  from 
five  to  ten  minutes  to  secure  homogeneity.  The  organism 
does  not  stain  by  Gram's  method.  It  may  be  colored  and 
examined  while  alive;  thus,  Cornil  and  Babes,  in  demon- 
strating it  in  the  rice-water  discharges,  "spread  out  one 
of  the  white  mucous  fragments  upon  a  glass  slide  and  allow 
it  to  dry  partially;  a  small  quantity  of  an  exceedingly 


Fig.    210. — Cover-glass  preparation  of   a  mucous  floccule  in   Asiatic 
cholera.     X  650  (Vierordt). 


weak  solution  of  methyl  violet  in  distilled  water  is  then 
applied  to  it,  and  it  is  flattened  out  by  pressing  down 
a  cover-glass,  over  which  is  placed  a  fragment  of  filter 
paper,  which  absorbs  any  excess  of  fluid  at  the  margin  of 
the  cover-glass.  The  characteristics  of  comma  bacilli  so 
prepared  and  examined  with  an  oil-immersion  lens  (X  700- 
800)  are  readily  made  out  because  of  the  slight  stain  they 
take  up,  and  because  they  still  retain  the  power  of  vigor- 
ous movement,  which  would  be  entirely  lost  if  the  specimen 
were  dried,  stained,  and  mounted  in  the  ordinary  fashion." 

Isolation  of  the  Organism.— One  of  the  best  methods 
of  securing  a  pure  culture  of  the  cholera  spirillum,  and  also 


Cultivation 


609 


of  making  a  bacteriologic   diagnosis  of  the   disease  in   a 
suspected  case,  is  probably  that  of  Schottelius. 

A  small  quantity  of  the  fecal  matter  is  mixed  with  bouillon  and 
stood  in  an  incubating  oven  for  twenty-four  hours.  If  the  cholera 
spirilla  are  present  they  will  grow  most  rapidly  at  the  surface  of  the 
liquid  where  the  supply  of  air  is  good.  A  pellicle  will  be  formed,  a 
drop  from  which,  diluted  in  melted  gelatin  and  poured  upon  plates, 
will  show  typical  colonies. 

Cultivation. — The  cholera  organism  is  easily  cultivated, 
and  grows  luxuriantly  upon  the  usual  laboratory  media. 


Fig.  211. — Spirillum  of     Asiatic  cholera;  colonies  two  days  old  upon 
a  gelatin  plate.     X  35  (Heim). 


Colonies. — The  colonies  grown  upon  gelatin  plates  are 
characteristic  and  appear  in  the  lower  strata  of  the  gelatin 
as  small  white  dots,  which  gradually  grow  out  to  the  surface, 
effect  a  slow  liquefaction  of  the  medium,  and  then  appear 
to  be  situated  in  little  pits  with  sloping  sides  (Fig.  211). 
This  appearance  suggests  that  the  plate  is  full  of  little  holes 
or  air-bubbles,  and  is  due  to  the  slow  evaporation  of  the 
liquefied  gelatin. 

Under  the  microscope  the  colony  of  the  cholera  spirillum 
is  fairly  well  characterized.  The  little  colonies  that  have 
39 


6io 


Asiatic  Cholera 


not  yet  reached  the  surface  of  the  gelatin  soon  show  a 
pale  yellow  color  and  an  irregular  contour.  They  are 
coarsely  granular,  the  largest  granules  being  in  the  center. 
As  the  colony  increases  in  size  the  granules  do  the  same 
and  attain  a  peculiar  transparent  character  suggestive  of 
powdered  glass.  The  slow  liquefaction  causes  the  colony  to 
be  surrounded  by  a  transparent  halo.  As  the  liquefied 
gelatin  evaporates,  the  colony  begins  to  sink,  and  also  to 
take  on  a  peculiar  rosy  color. 

Gelatin. — In  puncture  cultures  in  gelatin  the  growth  is 


Fig.  212. — Spirillum  cholerae  asiaticae;  gelatin  puncture  cultures  aged 
forty-eight  and  sixty  hours  (Shakespeare). 


again  quite  characteristic  (Fig.  212).  It  occurs  along 
the  entire  puncture,  best  at  the  surface,  where  it  is  in 
contact  with  the  atmosphere.  Liquefaction  of  the  medium 
begins  almost  at  once,  keeps  pace  with  the  growth,  but 
is  always  more  marked  at  the  surface  than  lower  down. 
The  result  of  this  is  the  formation  of  a  short,  rather  wide 
funnel  at  the  top  of  the  puncture.  As  the  growth  continues, 
evaporation  of  the  medium  takes  place  slowly,  so  that  the 
liquefied  gelatin  is  lower  than  the  solid  surrounding  portions, 
and  the  growth  appears  to  be  surmounted  by  an  air-bubble. 
The  luxuriant  development  of  the  spirilla  in  the  liquefying 


Vital  Resistance  611 

gelatin  is  followed  by  the  formation  of  considerable  sediment 
in  the  lower  third  or  half  of  the  liquefied  area.  This  solid 
material  consists  of  masses  of  spirilla  which  have  probably 
completed  their  life-cycle  and  become  inactive.  Under  the 
microscope  they  exhibit  the  most  varied  involution-forms. 
The  liquefaction  reaches  the  sides  of  the  tube  in  from  five 
to  seven  days,  but  is  not  complete  for  several  weeks. 

Agar-agar. — When  planted  upon  the  surface  of  agar- 
agar  the  spirilla  produce  a  grayish-white,  shining,  translu- 
cent growth  along  the  entire  line  of  inoculation.  It  is  in 
no  way  peculiar  or  characteristic.  The  vitality  of  the 
organism  is  retained  much  better  upon  agar-agar  than  upon 
gelatin,  and,  according  to  Frankel,  the  organism  can  be 
transplanted  and  grown  when  nine  months  old. 

Blood-serum. — The  growth  upon  blood-serum  is  also 
without  distinct  peculiarities;  gradual  liquefaction  of  the 
medium  occurs. 

Potato. — Upon  potato  the  spirilla  grow  well,  even  when 
the  reaction  is  acid.  In  the  incubator,  at  a  temperature  of 
37°  C.,  a  transparent,  slightly  brownish  or  yellowish-brown 
growth,  somewhat  resembling  that  of  glanders,  is  produced. 
It  contains  large  numbers  of  long  spirals. 

Bouillon. — In  bouillon  and  in  peptone  solution  the 
cholera  organisms  grow  well,  especially  upon  the  surface, 
where  a  folded,  wrinkled  pellicle  is  formed,  the  culture 
fluid  remaining  clear. 

Milk. — In  milk  the  growth  is  luxuriant,  but  does 
not  visibly  alter  its  appearance.  The  existence  of  cholera 
organisms  in  milk  is,  however,  rather  short-lived,  for  the 
occurrence  of  acidity  at  once  destroys  them. 

Vital  Resistance. — Although  an  organism  that  multiplies 
with  great  rapidity  under  proper  conditions,  the  cholera 
spirillum  does  not  possess  much  resisting  power.  Stern- 
berg  found  that  it  was  killed  by  exposure  to  52°  C.  for 
four  minutes,  but  Kitasato  found  that  ten  or  fifteen 
minutes'  exposure  to  55°  C.  was  not  always  fatal  to  it.  In 
a  moist  condition  the  organism  may  retain  its  vitality  for 
months,  but  it  is  very  quickly  destroyed  by  desiccation,  as 
was  found  by  Koch,  who  observed  that  when  dried  in  a  thin 
film  its  power  to  grow  was  destroyed  in  a  few  hours.  Kita- 
sato found  that  upon  silk  threads  the  vitality  might  be 
retained  longer.  Abel  and  Claussen  *  have  shown  that  it 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Jan.  31,  1895,  vol.  xvn,  No.  4. 


612  Asiatic  Cholera 

does  not  live  longer  than  twenty  or  thirty  days  in  fecal 
matter,  and  often  disappears  in  from  one  to  three  days. 
The  organism  is  very  susceptible  to  the  influence  of  carbolic 
acid,  bichlorid  of  mercury,  and  other  germicides,  and  is 
also  destroyed  by  acids.  Hashimoto  *  found  that  it  could 
not  live  longer  than  fifteen  minutes  in  vinegar  containing 
2.2-3.2  per  cent,  of  acetic  acid. 

According  to  Frankel,  in  eight  weeks  the  organisms  in 
the  liquefied  cultures  all  die,  and  cannot  be  transplanted. 
Kitasato,  however,  has  found  them  living  and  active  on 
agar-agar  after  from  ten  to  thirty  days,  and  Koch  was 
able  to  demonstrate  their  vitality  after  two  years. 

This  low  vital  resistance  of  the  microbe  is  very  fortunate, 
for  it  enables  us  to  establish  satisfactory  quarantine  for  the 
prevention  of  the  spread  of  the  disease.  Excreta,  soiled 
clothing,  etc.,  are  readily  rendered  harmless  by  the  proper 
use  of  disinfectants.  Water  and  food  are  rendered  innocu- 
ous by  boiling  or  cooking.  Vessels  may  be  disinfected  by 
thorough  washing  with  jets  of  boiling  water  discharged 
through  a  hose  connected  with  a  boiler,  and  baggage  can 
be  sterilized  by  superheated  steam. 

Metabolic  Products. — Indol  is  one  of  the  characteristic 
metabolic  products  of  the  cholera  spirillum.  As  the  cholera 
organisms  also  produce  nitrites,  all  that  is  necessary  to 
demonstrate  its  presence  in  a  colorless  solution  is  to  add  a 
drop  or  two  of  chemically  pure  sulphuric  acid,  when  the 
well-known  reddish  color  will  appear. 

The  organism  also  produces  acid  in  milk  and  other 
media.  Bitter  has  also  shown  that  the  cholera  organism 
produces  a  peptonizing  and  probably  also  a  diastatic 
ferment. 

Toxic  Products.— Rietsch  thinks  the  intestinal  changes 
depend  upon  the  action  of  the  peptonizing  ferment.  Can- 
tani,  Nicati  and  Rietsch,  Van  Ermengem,  Klebs,  and  others 
found  toxic  effects  from  cultures  administered  to  dogs 
and  other  animals.  Several  toxic  metabolic  products  of 
the  spirilla  have  been  isolated.  Brieger,  f  Brieger  and 
Frankel,  {  Gamaleia,  §  Sobernheim,  ||  and  Villiers  have 

*  "Kwai  Med.  Jour.,"  Tokyo,  1893. 

f  "Berliner  klin.  Wochenschrift,"  1887,  p.  817. 

J  "  Untersuchungen  iiber  die  Bakteriengifte,"  etc.,  Berlin,  1890. 

§  "Archiv  de  Med.  exp.,"  iv,  No.  2. 

||  "Zeitschrift  fur  Hygiene,"  xiv,  145,  1893. 


Pathogenesis  613 

studied  more  or  less  similar  toxic  products.  The  real  toxic 
substance  is,  however,  not  known. 

Pathogenesis. — Through  what  activity  the  cholera  or- 
ganism provokes  its  pathogenic  action  is  not  yet  deter- 
mined. The  organisms,  however,  abound  in  the  intestinal 
contents,  penetrate  sparingly  into  the  tissues,  but  slightly 
invade  the  lymphatics,  and  almost  never  enter  the  circula- 
tion ;  hence  it  is  but  natural  to  conclude  that  the  first  ac- 
tion must  be  an  irritative  one  depending  upon  toxin-for- 
mation in  the  intestine. 

In  the  beginning  of  the  disease  the  small  and  large  intes- 
tines are  deeply  congested,  almost  velvety  in  appearance, 
and  contain  liquid  fecal  matter.  The  patient  suffers  from 
diarrhea,  by  which  the  feces  are  hurried  on  and  be- 
come extremely  thin  from  the  admixture  of  a  copious 
watery  exudate.  As  the  feces  are  hurried  out,  more  and 
more  of  the  aqueous  exudate  accumulates,  until  the  intes- 
tine seems  to  contain  only  watery  fluid.  The  solitary 
glands  and  Peyer's  patches  are  found  enlarged  and  the 
mucosa  becomes  macerated  and  necrotic,  its  epithelium 
separating  in  small  shreds  or  flakes.  The  evacuations  of 
watery  exudate  rich  in  these  shreds  constitute  the  char- 
acteristic "rice-water  discharges"  of  the  disease.  As  the 
disease  progresses,  the  denudation  of  tissue  results  in  the 
formation  of  good-sized  ulcerations.  Perforations  and  deep 
ulcerations  are  rare.  Pseudo-membranous  formations  not 
infrequently  occur  upon  the  abraded  and  ulcerated  surfaces. 
The  other  mucous  membranes  of  the  alimentary  apparatus 
become  congested  and  abraded;  the  parenchyma  of  the 
liver,  kidneys,  and  other  organs  becomes  markedly  de- 
generated, so  that  the  urine  becomes  highly  albuminous 
and  very  scanty  in  consequence  of  the  anhydremia.  The 
cardio-vascular,  nervous,  and  respiratory  systems  present 
no  characteristic  changes. 

So  far  as  is  known,  cholera  is  a  disease  of  human  beings  only, 
and  never  occurs  spontaneously  in  the  lower  animals. 

Intraperitoneal  injection  of  the  virulent  cultures  produces 
fatal  peritonitis  in  guinea-pigs. 

Supposing  that  the  lower  animals  were  immune  against 
cholera  because  of  the  acidity  of  the  gastric  juice,  Nicati 
and  Rietsch,*  Van  Ermengem,  and  Kochf  have  suggested 
methods  by  which  the  micro-organisms  can  be  introduced 

*"Deutsch.  med.  Wochenschrift,"  1884. 
t  Ibid.,  1885. 


6 14  Asiatic  Cholera 

directly  into  the  intestine.  The  first-named  investigators 
ligated  the  common  bile-duct  of  guinea-pigs,  and  then 
injected  the  spirilla  into  the  duodenum  with  a  hypodermic 
needle,  with  the  result  that  the  animals  usually  died,  some- 
times with  choleraic  symptoms.  The  excessively  grave 
nature  of  the  operation  upon  such  a  small  and  delicately 
constituted  animal  as  a  guinea-pig,  however,  greatly  lessens 
the  value  of  the  experiment.  Koch's  method  of  infection 
by  the  mouth  is  much  more  satisfactory.  By  injecting 
laudanum  into  the  abdominal  cavity  of  guinea-pigs  the 
peristaltic  movements  of  the  intestine  can  be  checked.  The 
amount  necessary  for  the  purpose  is  large  and  amounts  to 
about  i  gram  for  each  200  grams  of  body-weight.  It 
completely  narcotizes  the  animals  for  a  short  time  (one  to 
two  hours),  but  they  recover  without  injury.  The  contents 
of  the  stomach  are  neutralized  after  administering  the 
opium,  by  introducing  5  c.c.  of  a  5  per  cent,  aqueous  solution 
of  sodium  carbonate  through  a  pharyngeal  catheter.  With 
the  gastric  contents  thus  alkalinized  and  the  peristalsis 
paralyzed,  a  bouillon  culture  of  the  cholera  spirillum  is 
introduced  through  the  stomach-tube.  The  animal  recovers 
from  the  manipulation,  but  shows  an  indisposition  to  eat, 
is  soon  observed  to  be  weak  in  the  posterior  extremities, 
subsequently  is  paralyzed,  and  dies  within  forty-eight  hours. 
The  autopsy  shows  the  intestine  congested  and  filled  with 
a  watery  fluid  rich  in  spirilla — an  appearance  which  Frankel 
declares  to  be  exactly  that  of  cholera.  In  man,  as  well 
as  in  these  artificially  infected  animals,  the  spirilla  are 
never  found  in  the  blood  or  tissues,  but  only  in  the  intestine, 
where  they  frequently  enter  between  the  basement  mem- 
brane and  the  epithelial  cells,  and  aid  in  the  detachment  of 
the  latter. 

Issaeff  and  Kolle  found  that  when  virulent  cholera  spirilla 
are  injected  into  the  ear- veins  of  young  rabbits  the  animals 
die  on  the  following  day  with  symptoms  resembling  the 
algid  state  of  human  cholera.  The  autopsy  in  these  cases 
showed  local  lesions  of  the  small  intestine  very  similar  to 
those  observed  in  cholera  in  man. 

Guinea-pigs  are  also  susceptible  to  intraperitoneal  injec- 
tions of  the  spirillum,  and  speedily  succumb.  The  symp- 
toms are  rapid  fall  of  temperature,  tenderness  over  the 
abdomen,  and  collapse.  The  autopsy  shows  an  abundant 
fluid  exudate  containing  the  micro-organisms,  and  injection 
and  redness  of  the  peritoneum  and  viscera. 


Detection  of  the  Organism  615 

Specificity. — The  cholera  spirillum  is  present  in  the  de- 
jecta of  cholera  with  great  regularity,  and  as  regularly  absent 
from  the  dejecta  of  healthy  individuals  and  those  suffering 
from  other  diseases.  There  is  no  satisfactory  proof  of  the 
specific  nature  of  the  organisms  to  be  obtained  by  experi- 
mentation upon  animals.  Animals  are  never  affected  by 
any  disease  similar  to  cholera  during  epidemics,  nor  do  foods 
mixed  with  cholera  discharges  or  with  pure  cultures  of  the 
cholera  spirillum  affect  them.  Subcutaneous  inoculations 
do  not  produce  cholera. 

Detection  of  the  Organism. — It  often  becomes  a  matter 
of  importance  to  detect  the  cholera  spirilla  in  drinking- 
water,  and,  as  the  number  in  which  the  bacteria  exist  in 
such  a  liquid  may  be  very  small,  difficulty  may  be  experienced 
in  finding  them  by  ordinary  methods.  One  of  the  most 
expeditious  methods  is  that  recommended  by  LofHer,  who 
adds  200  c.c.  of  the  water  to  be  examined  to  10  c.c.  of 
bouillon,  allows  the  mixture  to  stand  in  an  incubator  for 
from  twelve  to  twenty-four  hours,  and  then  makes  plate 
cultures  from  ^the  superficial  layer  of  the  liquid,  where,  if 
present,  the  development  of  the  spirilla  will  be  most  rapid 
because  of  the  free  access  of  air. 

Gordon  *  employs  a  medium  composed  of  Lemco  i  gram, 
peptone  i  gram,  sodium  bicarbonate  o.  i  gram,  starch  i 
gram,  and  distilled  water  100  c.c.  for  the  differentiation 
of  the  cholera  and  Finkler-Prior  spirilla.  If  the  medium 
be  tinted  with  litmus  and  the  cultures  grown  at  37°  C., 
a  strongly  acid  change  is  produced  by  the  true  cholera 
organism  in  twenty-four  hours.  The  Finkler-Prior  spirillum 
produces  but  slight  acidity,  which  first  appears  about  the 
third  day. 

The  identification  of  the  cholera  spirillum,  and  its  differen- 
tiation from  spiral  organisms  of  similar  morphology  obtained 
from  feces  or  water  in  which  no  cholera  organisms  are 
expected,  is  becoming  less  and  less  easy  as  our  knowledge 
of  the  organisms  increases.  The  following  points  may  be 
taken  into  consideration:  (i)  The  typical  morphology. 
The  true  cholera  organism  is  short,  has  a  single  curve,  is 
rounded  at  the  ends,  and  possesses  a  single  flagellum.  (2) 
The  infectivity.  Freshly  isolated  cultures  should  be  patho- 
genic for  guinea-pigs  and  harmless  to  pigeons.  (3)  Vegeta- 

*  "British  Medical  Journal,"  July  28,  1906. 


616  Asiatic  Cholera 

live:  The  organism  should  liquefy  10  per  cent,  gelatin  and 
should  not  coagulate  milk.  (4)  Metabolic:  Indol  reaction 
should  be  marked.  (5)  Immunity  reactions:  The  organism 
when  injected  into  guinea-pigs  in  ascending  doses  should 
occasion  immunity  against  the  typical  cholera  organism, 
and  the  serum  of  the  immunized  guinea-pig,  when  intro- 
duced into  a  new  guinea-pig,  should  protect  it  from  infection 
and  produce  Pfeiffer's  phenomenon.  The  blood-serum  of 
animals  immunized  against  the  cholera  organism  should 
agglutinate  the  doubtful  organism  in  approximately  the 
same  dilution,  and  that  of  animals  immunized  to  the  doubtful 
organism  should  agglutinate  the  cholera  organism  recipro- 
cally. Both  organisms  should  have  equal  capacity  for 
absorbing  complements  and  amboceptors  from  blood-serum. 
(6)  The  true  cholera  organism  should  not  be  hemolytic. 
Too  much  reliance  must  not  be  placed  upon  the  agglu- 
tination tests  alone,  as  will  be  made  clear  by  a  perusal  of 
the  paper  upon  Bacteriological  Diagnosis  of  Cholera  by 
Ruffer.* 

Immunity. — One  attack  of  cholera  usually  leaves  the  vic- 
tim immune  from  further  attacks  of  the  disease.  Gruber 
and  Wiener,!  Haffkine, }  Pawlowsky, §  and  Pfeiffer||  have  im- 
munized animals  against  toxic  substances  from  cholera  cul- 
tures or  against  living  cultures. 

Pfeiffer  and  Vogedes**  have  applied  the  "immunity  re- 
action" to  the  differentiation  of  cholera  spirilla  in  cultures. 
A  hanging  drop  of  a  i  :  50  mixture  of  a  powerful  anticholera 
serum  and  a  particle  of  cholera  culture  is  made  and  ex- 
amined under  the  microscope.  The  cholera  spirilla  at  once 
become  inactive,  and  are  in  a  short  time  converted  into 
little  rolled-up  masses.  If  the  culture  added  be  a  spirillum 
other  than  the  true  cholera  spirillum,  instead  of  being 
destroyed  the  micro-organisms  multiply  and  thrive  in  the 
mixture  of  serum  and  bouillon. 

Sobernheimft  found  the  Pfeiffer  reaction  specific  against 

*  "British  Medical  Journal,"  March  30,  1907,  i,  p.  735. 
t  "Centralbl.  f.  Bakt.,"  etc.,  1892,  xiv,  p.  76. 

J"L,e  Bull,  med.,"  1892,  p.  1113,  and  "Brit.  Med.  Jour.,"  1893, 
p.  278. 

§  "Deutsche  med.  Wochenschrift,"  1893,  No.  22. 
||  "Zeitschrift  fur  Hygiene,"  Bd.  xvm  and  xx. 

**  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  March  21,  1896,  Bd.  xix, 
No.  1 1 . 

t|  "Zeitschrift  fur  Hygiene,"  xx,  p.  438. 


Serum  Therapy  and  Prophylaxis  617 

cholera  alone,  and  thought  the  protection  not  due  to  the 
strongly  bactericidal  property  of  the  serum,  but  to  its 
stimulating  effect  upon  the  body-cells;  for  if  the  serum  be 
heated  to  6o°-7o°  C.,  and  its  bactericidal  power  thus  de- 
stroyed, it  is  still  capable  of  producing  immunity;  This, 
of  course,  is  in  keeping  with  our  present  knowledge  of 
the  immune  body,  which  is  not  destroyed  by  such  tem- 
peratures. 

The  immunity  produced  by  the  injection  of  the  spirilla 
into  guinea-pigs  continues  in  some  cases  as  long  as  four  and 
a  half  months,  but  the  power  of  their  serum  to  confer 
immunity  is  lost  much  sooner. 

Serum  Therapy  and  Prophylaxis. — Of  the  numerous 
attempts  to  produce  immunity  against  cholera  in  man  or 
to  cure  cholera  when  once  established  in  the  human  organ- 
ism, nothing  very  favorable  can  be  said.  Experiments  in 
this  field  are  not  new.  As  early  as  1885  Ferran,  in  Spain, 
administered  hypodermic  injections  of  pure  virulent  cul- 
tures of  the  cholera  spirillum,  in  the  hope  of  bringing 
about  immunity.  The  work  of  Haffkine,*  however,  is  the 
chief  important  contribution,  and  his  method  seems  to  be 
followed  by  a  positive  diminution  of  mortality  in  protected 
individuals.  Haffkine  uses  two  vaccines — one  mild,  the 
other  so  powerful  that  it  would  bring  about  extensive  tissue- 
necrosis  and  perhaps  death  if  used  alone.  His  studies 
embrace  more  than  40,000  inoculations  performed  in  India. 
The  following  extract  will  show  results  obtained  in  1895: 

"1.  In  all  those  instances  where  cholera  has  made  a  large  number 
of  victims, — that  is  to  say,  where  it  has  spread  sufficiently  to  make 
it  probable  that  the  whole  population,  inoculated  and  uninoculated, 
were  equally  exposed  to  the  infection, — in  all  these  places  the  results 
appeared  favorable  to  inoculation. 

"2.  The  treatment  applied  after  an  epidemic  actually  breaks  out 
tends  to  reduce  the  mortality  even  during  the  time  which  is  claimed 
for  producing  the  full  effect  of  the  operation.  In  the  Goya  Garl, 
where  weak  doses  of  a  relatively  weak  vaccine  had  been  applied,  this 
reduction  was  to  half  the  number  of  deaths ;  in  the  coolies  of  the  Assam- 
Burmah  survey  party,  where,  as  far  as  I  can  gather  from  my  pre- 
liminary information,  strong  doses  have  been  applied,  the  number 
of  deaths  was  reduced  to  one-seventh.  This  fact  would  justify  the 
application  of  the  method  independently  of  the  question  as  to  the 
exact  length  of  time  during  which  the  effect  of  this  vaccination  lasts. 

"3.  In  Lucknow,  where  the  experiment  was  made  on  small  doses 
of  weak  vaccines,  a  difference  in  cases  and  deaths  was  still  noticeable 
in  favor  of  the  inoculated  fourteen  to  fifteen  months  after  vaccination 

*"Le  Bull,  med.,"  1892,  p.  1113;  "Indian  Med.  Gazette,"  1893, 
p.  97;  "Brit.  Med.  Jour.,"  1893,  p.  278. 


618  Asiatic  Cholera 

in  an  epidemic  of  exceptional  virulence.  This  makes  it  probable 
that  a  protective  effect  could  be  obtained  even  for  long  periods  ol 
time  if  larger  doses  of  a  stronger  vaccine  were  used. 

"4.  The  best  results  seem  to  be  obtained  from  application  of  middle 
doses  of  both  anticholera  vaccines,  the  second  one  being  kept  at  the 
highest  possible  degree  of  virulence  obtainable. 

"5.  The  most  prolonged  observations  on  the  effect  of  middle  doses 
were  made  in  Calcutta,  where  the  mortality  from  the  eleventh  up  to 
the  four  hundred  and  fifty-ninth  day  after  vaccination  was,  among 
the  inoculated,  17.24  times  smaller,  and  the  number  of  cases  19.27 
times  smaller  than  among  the  not  inoculated." 

Pawlowsky  and  others  have  found  the  dog  susceptible  to 
cholera,  and  have  utilized  it  in  the  preparation  of  an  anti- 
toxic serum.  The  dogs  were  first  immunized  against 
attenuated  cultures,  then  against  more  and  more  virulent 
cultures,  until  a  serum  was  obtained  whose  value  was 
estimated  at  i  :  130.000  upon  experimental  animals. 

Freymuth  *  and  others  have  endeavored  to  secure  favor- 
able results  from  the  injection  of  blood-serum  from  con- 
valescent patients  into  the  diseased.  One  recovery  out 
of  three  cases  treated  is  recorded. 

In  all  these  preliminaries  the  foreshadowing  of  a  future 
therapeusis  must  be  evident,  but  as  yet  nothing  satis- 
factory has  been  achieved. 

One  of  the  chief  errors  made  in  the  experimental  prepara- 
tion of  anticholera  serums  is  that  efforts  have  been  directed 
toward  endowing  the  blood  with  the  power  of  resisting 
and  destroying  the  bacteria  that  rarely,  if  ever,  reach  it. 
The  two  essentials  to  be  aimed  at  are  an  antitoxin  to  neu- 
tralize the  depressing  effects  of  the  toxalbumin,  and  some 
means  of  destroying  the  bacteria  in  the  intestine. 

The  cholera  spirillum  is  one  of  a  considerable-sized  group 
of  closely  related  organisms,  from  some  of  which  it  is  dif- 
ferentiated with  difficulty. 

Sanitation. — The  first  appearance  of  cholera  may  depend 
upon  the  introduction  of  the  micro-organisms  upon  fomites, 
hence  to  avoid  epidemics  it  is  necessary  to  disinfect  all  such 
coming  from  cholera  infected  localities.  Especially  are  food- 
stuffs and  particularly  raw  vegetables  and  fruits  to  be  con- 
sidered. All  such  should  either  be  avoided  or  only  eaten  after 
cooking. 

So  soon  as  cholera  asserts  itself,  the  chief  danger  lies  in  the 
probable  contamination  of  the  water-supply.  To  prevent 
this  the  utmost  effort  must  be  made  to  locate  all  cases  and  see 

*  "Deutsche  med.  Wochenschrift,"  1893,  No.  43. 


Cultivation  619 

that  the  dejecta  are  thoroughly  disinfected,  and  as  the  micro- 
organisms persist  in  the  intestinal  discharges  for  some  weeks 
after  convalescence,  the  patients  should  not  too  soon  be  dis- 
charged from  the  hospital,  but  should  be  retained  until  a 
bacteriologic  examination  shows  no  more  comma  bacilli  in 
the  feces.  During  an  epidemic  the  water  consumed  should  all 
be  boiled,  raw  milk  should  be  avoided,  and  no  green  or  un- 
cooked vegetables  or  fruits  eaten.  Foods  should  be  carefully 
defended  from  flies,  which  may  carry  the  organisms  to  them 
and  infect  them.  The  intestinal  evacuations  and  all  the 
clothing,  bedding,  and  other  articles  used  by  the  patients 
should  be  carefully  disinfected. 

SPIRILLA  RESEMBLING  THE  CHOLERA  SPIRILLUM. 
THE  FINKLER  AND   PRIOR  SPIRILLUM    (VIBRIO  PROTEUS). 

Similar  in  morphology  to  the  spirillum  of  cholera,  and  in 
other  respects  closely  related  to  it,  is  the  spirillum  obtained 
from  the  feces  of  a  case  of  cholera  nostras  by  Finkler  and 
Prior.* 

Morphology. — It  is  shorter  and  stouter,  with  a  more 
pronounced  curve  than  the  cholera  spirillum,  and  rarely 
forms  long  spirals.  The  central  portion  is  also  somewhat 
thinner  than  the  ends,  which  are  a  little  pointed  and  give 
the  organism  a  less  uniform  appearance.  Involution 
forms  are  common  in  cultures,  and  appear  as  spheres, 
spindles,  clubs,  etc.  Like  the  cholera  spirillum,  each  organ- 
ism is  provided  with  a  single  flagellum  situated  at  its  end, 
and  is  actively  motile. 

Staining. — The  organism  stains  readily  with  the  ordinary 
solutions,  but  not  by  Gram's  method. 

Cultivation.  —  Colonies.  —  The  growth  upon  gelatin 
plates  is  rapid,  and  leads  to  such  extensive  liquefaction 
that  four  or  five  dilutions  must  frequently  be  made  to  se- 
cure few  enough  organisms  to  enable  one  to  observe  the 
growth  of  a  single  colony.  To  the  naked  eye  the  deep  colo- 
nies appear  as  small  white  points.  They  rapidly  reach  the 
surface,  begin  liquefaction  of  the  gelatin,  and  by  the  sec- 
ond day  appear  about  the  size  of  lentils,  and  are  situated 
in  little  depressions.  Under  the  microscope  they  are 
yellowish  brown,  finely  granular,  and  are  surrounded  by 

*"Centralbl.  fur  allg.  Gesundheitspflege,"  Bd.  I,  Bonn,  1885; 
"Deutsche  med.  Wochenschrift,"  1884,  p.  632. 


620  The  Finkler  and  Prior  Spirillum 

a  zone  of  sharply  circumscribed  liquefied  gelatin.  Careful 
examination  with  a  high-power  lens  shows  rapid  movement 
of  the  granules  in  the  colony. 

Gelatin  Punctures. — In  gelatin  punctures  the  growth 
takes  place  rapidly  along  the  whole  length  of  the  puncture, 
forming  a  stocking-shaped  liquefaction  filled  with  cloudy 
fluid  which  does  not  precipitate  rapidly;  a  rather  smeary, 
whitish  scum  is  usually  formed  upon  the  surface.  The 
more  extensive  and  more  rapid  the  liquefaction  of  the 
medium,  the  wider  the  top  to  the  funnel,  the  absence  of  the 
air-bubble,  and  the  clouded  nature  of  the  liquefied  material, 


^HP** 


Fig.  213. — Spirillum  of  Finkler  and  Prior,  from  an  agar-agar  culture. 
X  1000  (Itzerott  and  Niemann). 

all  serve  to  differentiate  the  culture  from  the  cholera  spiril- 
lum. 

Agar-agar. — Upon  agar-agar  the  growth  is  also  rapid, 
and  in  a  short  time  the  whole  surface  of  the  culture  medium 
is  covered  with  a  moist,  thick,  slimy  coating,  which  may 
have  a  slightly  yellowish  tinge. 

Bouillon. — In  bouillon  the  organism  causes  a  diffuse 
turbidity  with  a  more  or  less  distinct  pellicle  on  the  surface. 
In  sugar-containing  culture  media  it  causes  no  fermenta- 
tion and  generates  no  gas. 

Potato. — The  cultures  upon  potato  are  also  different 
from  those  of  the  cholera  organism,  for  the  Finkler  and  Prior 
spirilla  grow  rapidly  at  the  room  temperature,  and  produce 


Cultivation 


621 


Fig.  214. — Spirillum  of  Finkler  and  Prior;  colony  twenty-four  hours 
old,  as  seen  upon  a  gelatin  plate.     X  100  (Frankel  and  Pfeiffer). 


Fig.  215. — Spirillum  of  Finkler  and  Prior;  gelatin  puncture  cultures 
aged  forty-eight  and  sixty  hours  (Shakespeare). 


622  Spirillum  of  Denecke 

a  grayish-yellow,  slimy  shining  layer,  which  may  cover  the 
whole  of  the  culture  medium. 

Blood-serum. — Blood-serum  is  rapidly  liquefied  by  the 
organism. 

The  spirillum  does  not  grow  well  in  milk,  and  speedily  dies 
in  water. 

Metabolic  Products. — The  organism  does  not  produce 
indol.  Buchner  has  shown  that  in  media  containing  some 
glucose  an  acid  reaction  is  produced.  Proteolytic  enzymes 
capable  of  dissolving  gelatin,  blood-serum,  and  casein  are 
formed. 

Pathogenesis. — It  was  at  first  supposed  that  if  not  the 
spirillum  of  cholera  itself,  this  was  a  very  closely  allied 
organism.  Later  it  was  supposed  to  be  the  cause  of  cholera 
nostras.  At  present  it  is  a  question  whether  the  organism 
has  any  pathologic  significance.  It  was  in  one  case  secured 
by  Knisl  from  the  feces  of  a  suicide,  and  has  been  found  in 
carious  teeth  by  Miiller. 

When  injected  into .  the  stomach  of  guinea-pigs  treated 
with  tincture  of  opium  according  to  the  method  of  Koch, 
about  30  per  cent,  of  the  animals  die,  but  the  intestinal 
lesions  produced  are  not  identical  with  those  produced  by 
the  cholera  spirillum.  The  intestines  in  such  cases  are  pale 
and  filled  with  watery  material  having  a  strong  putrefactive 
odor.  This  fluid  teems  with  the  spirilla. 

It  seems  unlikely,  from  the  evidence  thus  far  collected, 
that  the  Kinkier  and  Prior  spirillum  is  pathogenic  for  the 
human  species.  As  Krankel  points  out,  it  is  probably  a 
frequent  and  harmless  inhabitant  of  the  human  intestine. 

THE  SPIRILLUM  OF  DENECKE  (VIBRIO  TYROGENUM). 

Another  organism  with  a  partial  resemblance  to  the 
cholera  spirillum  was  found  by  Denecke  *  in  old  cheese. 

Morphology. — Its  form  is  similar  to  that  of  the  cholera 
spirillum,  the  shorter  individuals  being  of  equal  diameter 
throughout.  The  spiral  forms  are  longer  than  those  of  the 
Kinkier  and  Prior  spirillum,  and  are  more  tightly  coiled  than 
those  of  the  cholera  spirillum. 

Like  its  related  species,  tLis  micro-organism  is  actively 
motile  and  possesses  a  terminal  flagellum. 

*  "Deutsche  med.  Wochenschrift,"  1885. 


Cultivation  623 

Cultivation. — It  grows  at  the  room  temperature,  as  well 
as  at  37°  C.,  in  this  respect,  as  in  its  reaction  to  stains,  much 
resembling  the  other  two. 

Colonies. — Upon  gelatin  plates  the  growth  of  the  colonies 
is  much  more  rapid  than  that  of  the  cholera  spirillum, 
though  slower  than  that  of  the  Kinkier  and  Prior  spirillum. 
The  colonies  appear  as  small  whitish,  round  points,  which 


Fig.  216. — Spirillum  of  Denecke,  from  an  agar-agar  culture.      X  1000 
(Itzerott  and  Niemann). 

soon  reach  the  surface  of  the  gelatin  and  commence  lique- 
faction. By  the  second  day  each  is  about  the  size  of  a 
pin's  head,  has  a  yellow  color,  and  occupies  the  bottom  of 
a  conical  depression.  The  appearance  is  much  like  that 
of  colonies  of  the  cholera  spirillum. 

The  microscope  shows  the  colonies  to  be  of  irregular 
shape  and  coarsely  granular,  pale  yellow  at  the  edges, 
gradually  becoming  intense  toward  the  center,  and  at  first 
circumscribed,  but  later  surrounded  by  clear  zones,  resulting 
from  the  liquefaction  of  the  gelatin.  These,  according  to 
the  illumination,  appear  pale  or  dark.  The  colonies  differ 
in  appearance  from  those  of  cholera  in  the  prompt  lique- 
faction of  the  gelatin,  their  rapid  growth,  yellow  color, 
irregular  form,  and  distinct  lines  of  circumscription. 

Gelatin  Punctures. — In  gelatin  punctures  the  growth 
takes  place  all  along  the  track  of  the  wire,  and  forms  a 
cloudy  liquid  which  precipitates  at  the  apex  in  the  form  of 


624  Spirillum  of  Denecke 

a  coiled  mass.  Upon  the  surface  a  delicate,  imperfect, 
yellowish  scum  forms.  Liquefaction  of  the  entire  gelatin 
generally  requires  about  two  weeks. 

Agar-agar. — Upon  agar-agar  this  spirillum  forms  a  thin 
yellowish  layer  which  spreads  quickly  over  most  of  the  sur- 
face. 

Bouillon. — In  bouillon  the  growth  of  the  organism  is 
characterized  by  a  diffuse  turbidity.  No  gas-formation 
occurs  in  sugar-containing  media. 


Fig.  217.— Spirillum  of  Denecke;  gelatin  puncture  cultures  aged  forty- 
eight  and  sixty  hours  (Shakespeare). 

Potatoes. — The  culture  upon  potato  is  luxuriant  if 
grown  in  the  incubating  oven.  It  appears  as  a  distinct 
yellowish,  moist  film,  and  when  examined  microscopically 
is  seen  to  contain  beautiful  long  spirals. 

Metabolic  Products. — The  organism  produces  no  indol. 

Pathogenesis. — The  spirillum  of  Denecke  is  mentioned 
only  because  of  its  morphologic  resemblance  to  the  cholera 
spirillum.  It  is  not  associated  with  any  human  disease. 
Experiments,  however,  have  shown  that  when  the  spirilla 
are  introduced  into  guinea-pigs  whose  gastric  contents  are 
alkalinized  and  whose  peristalsis  is  paralyzed  with  opium, 
about  20  per  cent,  of  the  animals  die. 


Cultivation  625 

THE  SPIRILLUM  OF  GAMAL£IA*  (VIBRIO  METSCHNIKOVI). 

Resembling  the  cholera  spirillum  in  morphology  and 
vegetation,  and  possibly,  as  has  been  suggested,  a  de- 
scendant of  the  same  original  stock,  is  a  spirillum  which 
Gamaleia  cultivated  from  the  intestines  of  chickens  affected 
with  a  disease  similar  to  chicken-cholera. 

Morphology. — This  spirillum  is  a  trifle  shorter  and 
thicker  than  the  cholera  spirillum.  It  is  a  little  more 


Fig.  2 1 8. — Spirillum  metschnikovi,  from  an  agar-agar  culture.     X  1000 
(Itzerott  and  Niemann) 


curved,  and  has  similar  rounded  ends.  It  forms  long  spirals 
in  appropriate  media,  and  is  actively  motile.  Bach  spi- 
rillum is  provided  with  a  terminal  flagellum.  No  spores 
have  been  demonstrated. 

Staining. — The  organism  stains  easily,  the  ends  more 
deeply  than  the  center.  It  is  not  stained  by  Gram's  method. 

Cultivation. — It  grows  well  both  at  the  temperature  of 
the  room  and  at  that  of  incubation. 

Colonies. — The  colonies  upon  gelatin  plates  have  a 
marked  resemblance  to  those  of  the  cholera  spirillum,  yet 
there  is  a  difference;  and  as  Pfeiffer  says,  "it  is  compara- 
tively easy  to  differentiate  between  a  plate  of  pure  cholera 
spirillum  and  a  plate  of  pure  Spirillum  metschnikovi,  yet  it 

*  "Ann.  de  1'Inst.  Pasteur,"  1888. 
40 


626  Spirillum  of  Gamaleia 

is  almost  impossible  to  pick  out  a  few  colonies  of  the  latter 
if  mixed  upon  a  plate  with  the  former." 

Frankel  regards  this  organism  as  a  species  intermediate 
between  the  cholera  and  the  Kinkier- Prior  spirilla. 

The  colonies  upon  gelatin  plates  appear  in  about  twelve 
hours  as  small  whitish  points,  and  rapidly  develop,  so  that 
by  the  end  of  the  third  day  large  saucer-shaped  liquefactions 
resembling  colonies  of  the  Finkler- Prior  spirilla  occur.  The 
liquefaction  of  the  gelatin  is  quite  rapid,  the  resulting  fluid 
being  turbid.  Usually,  upon  a  plate  of  Vibrio  metschnikovi 
some  colonies  are  present  which  closely  resemble  those  of 


Fig.  219.— Spirillum  metschnikovi;  puncture  culture  in  gelatin  forty- 
eight  hours  old  (Frankel  and  Pfeiffer). 

the  cholera  spirillum,  being  deeply  situated  in  conical  de- 
pressions in  the  gelatin.  Under  the  microscope  the  contents 
of  the  colonies,  which  appear  of  a  brownish  color,  are  ob- 
served to  be  in  rapid  motion.  The  edges  of  the  bacterial 
mass  are  fringed  with  radiating  organisms 

Gelatin  Punctures. — In  gelatin  tubes  the  growth  closely 
resembles  that  of  the  cholera  organism,  but  develops  more 
slowly. 

Agar-agar. — Upon  the  surface  of  agar-agar  a  yellowish- 
brown  growth  develops  along  the  whole  line  of  inoculation. 

Potato. — On  potato  at  the  room  temperature  no  growth 
occurs,  but  at  the  temperature  of  the  incubator  a  luxuriant 


Metabolic  Products  627 

yellowish-brown  growth  takes  place.  Sometimes  the  color 
is  quite  dark,  and  chocolate-colored  potato  cultures  are  not 
uncommon. 

Bouillon. — In  bouillon  the  growth  which  occurs  at  the 
temperature  of  the  incubator  is  quite  characteristic,  and 
very  different  from  that  of  the  cholera  spirillum.  The 
entire  medium  becomes  clouded,  of  a  grayish-white  color, 
and  opaque.  A  folded  and  wrinkled  pellicle  forms  upon  the 
surface. 

Milk. — When  grown  in  litmus  milk,  the  original  blue 
color  is  changed  to  pink  in  a  day,  and  at  the  end  of  another 
day  the  color  is  all  destroyed  and  the  milk  coagulated. 
Ultimately  the  clots  of  casein  sediment  in  irregular  masses, 
from  the  clear,  colorless  whey. 

Vital  Resistance. — The  organism,  like  the  cholera 
vibrio,  is  very  susceptible  to  the  influence  of  acids,  high 
temperatures,  and  drying.  The  thermal  death-point  is 
50°  C.,  continued  for  five  minutes. 

Metabolic  Products. — The  addition  of  sulphuric  acid  to 
a  culture  grown  in  a  medium  rich  in  peptone  produces  the 
same  rose  color  observed  in  cholera  cultures  and  shows  the 
presence  of  indol.  When  glucose  is  added  to  the  bouillon, 
no  fermentation  or  gas-production  results.  The  organism 
produces  acids  and  curdling  enzymes. 

Pathogenesis. — The  organism  is  pathogenic  for  animals, 
but  not  for  man.  Pfeiffer  has  shown  that  chickens  and 
guinea-pigs  are  highly  susceptible,  and  when  inoculated  under 
the  skin  usually  die.  The  virulent  organism  is  invariably 
fatal  for  pigeons.  W.  Rindfleisch  has  pointed  out  that  this 
constant  fatality  for  pigeons  is  a  valuable  criterion  for  the 
differentiation  of  this  spirillum  from  that  of  cholera,  as  the 
subcutaneous  injection  of  the  most  virulent  cholera  cultures 
is  never  fatal  to  pigeons,  the  birds  only  dying  when  the 
injections  are  made  into  the  muscles  in  such  a  manner  that 
the  muscular  tissue  is  injured  and  becomes  a  locus  minoris 
resistenti<B.  When  guinea-pigs  are  treated  by  Koch's 
method  of  narcotization  and  cholera  infection,  the  tem- 
perature of  the  animal  rises  for  a  short  time,  then  ab- 
ruptly falls  to  33°  C.  or  less.  Death  follows  in  from  twenty 
to  twenty-four  hours.  A  distinct  inflammation  of  the  intes- 
tine, with  exudate  and  numerous  spirilla,  may  be  found. 
The  spirilla  can  also  be  found  in  the  heart's  blood  and  in 
the  organs  of  such  guinea-pigs.  When  the  bacilli  are  intro- 


628  Vibrio  Schuylkiliensis 

duced  by  subcutaneous  inoculation,  the  autopsy  shows  a 
bloody  edema  and  a  superficial  necrosis  of  the  tissues. 

The  organisms  can  be  found  in  the  blood  and  all  the 
organs  of  pigeons  and  young'  chickens,  in  such  large  numbers 
that  Pfeiffer  has  called  the  disease  Vibrionensepticcemia.  In 
the  intestines  very  few  alterations  are  noticeable,  and  very 
few  spirilla  can  be  found. 

Immunity. — Gamaleia  has  shown  that  pigeons  and 
guinea-pigs  can  be  made  immune  by  inoculating  them  with 
cultures  sterilized  for  a  time  at  a  temperature  of  100°  C. 
Mice  and  rabbits  are  immune,  except  to  very  large  doses. 

VIBRIO  SCHUYLKILIENSIS  (ABBOTT). 

Morphology. — This  micro-organism,  closely  resembling 
the  cholera  spirillum,  was  found  by  Abbott  *  in  sewage- 
polluted  water  from  the  Schuylkill  River  at  Philadelphia. 

Cultivation. — Colonies. — The  colonies  developed  upon 
gelatin  plates  very  closely  resemble  those  of  the  Spirillum 
metschnikovi. 

Gelatin  Punctures. — In  gelatin  puncture  cultures  the 
appearance  is  exactly  like  the  true  cholera  spirillum.  At 
times  the  growth  is  a  little  more  rapid. 

Agar-agar. — The  growth  on  agar  is  luxuriant,  and  gives 
off  a  pronounced  odor  of  indol. 

Blood-serum. — Loffler's  blood-serum  is  apparently  not 
a  perfectly  adapted  medium,  but  upon  it  the  organisms 
grow,  with  resulting  liquefaction. 

Potato. — Upon  potato,  at  the  point  of  inoculation  a  thin, 
glazed,  more  or  less  dirty  yellow  growth,  shading  to  brown 
and  sometimes  surrounded  by  a  flat,  dry,  lusterless  zone,  is 
formed. 

Milk. — In  litmus  milk  a  reddish  tinge  develops  after 
the  milk  is  kept  twenty-four  hours  at  body-temperature. 
After  forty-eight  hours  this  color  is  increased  and  the  milk 
coagulates. 

Metabolic  Products. — In  peptone  solutions  indol  is 
easily  detected.  No  gas  is  produced  in  glucose-containing 
culture  media.  Acids  and  coagulating  enzymes  are  formed. 
The  organism  is  a  facultative  anaerobe. 

*  "Journal  of  Experimental  Medicine,"  vol.  I,  No.  3,  July,  1896, 
p.  419. 


Immunity  629 

Vital  Resistance. — The  thermal  death-point  is  50°  C. 
maintained  for  five  minutes. 

Pathogenesis. — The  organism  is  pathogenic  for  pigeons, 
guinea-pigs,  and  mice,  behav-ing  much  like  Spirillum 
metschnikovi.  No  Pfeiffer's  phenomenon  was  observed 
with  the  use  of  serum  from  immunized  animals. 

Immunity. — Immunity  could  be  produced  in  pigeons, 
and  it  was  found  that  the  serum  was  protective  against  both 
Vibrio  schuylkiliensis  and  Spirillum  metschnikovi,  the  im- 
munity thus  produced  being  of  about  ten  days'  duration. 

In  a  second  paper  by  Abbott  and  Bergey  *  it  was  shown 
that  the  vibrios  occurred  in  the  water  during  all  four  seasons 
of  the  year,  and  in  all  parts  of  the  river  within  the  city,  both 
at  low  and  at  high  tide.  They  were  also  found  in  the 
sewage  emptying  into  the  river,  and  in  the  water  of  the 
Delaware  River  as  frequently  as  in  that  of  the  Schuylkill. 

One  hundred  and  ten  pure  cultures  were  isolated  from 
the  sources  mentioned  and  subjected  to  routine  tests.  It 
was  found  that  few  or  none  of  them  were  identical  in  all 
points.  There  seems  to  be,  therefore,  a  family  of  river 
spirilla,  closely  related  to  one  another,  like  the  different 
colon  bacilli. 

The  opinion  expressed  is  that  "the  only  trustworthy 
difference  between  many  of  these  varieties  and  the  true 
cholera  spirillum  is  the  specific  reaction  with  serum  from 
animals  immune  against  cholera,  or  by  Pfeiffer's  method  of 
intraperitoneal  testing  in  such  animals." 

In  discussing  these  spirilla  of  the  Philadelphia  waters 
Bergey  f  says : 

' '  The  most  important  point  with  regard  to  the  occurrence 
of  these  organisms  in  the  river  water  around  Philadelphia 
is  the  fact  that  similar  organisms  have  been  found  in  the 
surface  waters  of  the  European  cities  in  which  there  had 
recently  been  an  epidemic  of  Asiatic  cholera,  notably  at 
Hamburg  and  Altona.  .  .  .  The  foremost  bacteriolo- 
gists of  Europe  have  been  inclined  to  the  opinion  that  the 
organisms  which  they  found  in  the  surface  waters  of  the 
European  cities  were  the  remains  of  the  true  cholera  organ- 
ism, and  that  the  deviations  in  the  morphologic  and  biologic 
characters  from  those  of  the  cholera  organism  were  brought 
about  by  their  prolonged  existence  in  water.  No  such 

*  "Journal  of  Experimental  Medicine,"  vol.  n,  No.  5,  p.  535. 
t  "Journal  Amer.  Med.  Assoc.,"  Oct.  23,  1897. 


630  Vibrio  Schuylkiliensis 

explanation  of  the  occurrence  of  the  organisms  in  Phila- 
delphia waters  can  be  given." 

A  number  of  interesting  spirilla,  more  or  less  closely 
resembling  that  of  Asiatic  cholera,  have  been  described 
from  time  to  time.  Their  variation  from  the  true  cholera 
organism  can  best  be  determined  by  an  examination  of  the 
following  table,  though  for  precise  information  the  student 
will  do  well  to  look  up  the  original  descriptions,  references 
to  which  are  given  in  each  case. 


tZ=3?*?* 

COC/5C/)C/2C/5CCtflC/5t/5CDCfl 

•O'O'O'C'O'O'OXJ'U'O'O 

U3C/2      CflC/5 

•CT3  y-UTJ 

n>s  o 

sxil?= 

ccccccccccc 
33==53==333 

c  c  ITJS  c 

3   3   -t  3   3 

icr  klin.  Wochenschrift," 
de  1'Inst.  Pasteur,"  t.  n, 
iv  fiir  Hygiene,"  xxi,  189 
albl.  f.  Bakt.,"  etc.,  Bd. 
jienische  Rundschau,"  18 

dunbarensis  (Dunbar  ||) 
danubicus  (Heider**) 
I  (Wernickett)  •  •  • 
II  (Wernickett)  -  -  . 
liquefaciens  (Bonhoff  g§ 
weibeli  (Weibel  ||||)  .  . 
milleri  (Miller***)  .  . 
terrigenus  (Giintherttt 
berolinensis  (Neisser|t^ 
aquatilis  (Guntherggg) 
schuylkiliensis  (Abbott 

Intestinal  Orou 

cholerae  asiaticae  (Koch 
i  choleras  nostras  (Vibrio 
orf)  
tyrogenum  (Denecke  t) 

metschnikovi  (Gamaleif 
Water  Group. 

S  J*  -   °°  " 

C.       >  —  ' 

^      .   XJ3? 

«  "O  j»  88 

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m        3*t~=~"= 

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Found  in  Water. 

1    #87 

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Stain  by  Gram's 
Method. 

2.       C   3-t/i  x 

rr  3-o  o 

-i-  T  H-  -f  -f  +  +  -i-  -f  +  + 

+  +  -T          -t- 

Comma  Shape. 

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Thick  Spirals. 

5  !*ll 

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Slender  Spirals. 

5  I5&S- 

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0  C   0         0 

Spores. 

1  -INs- 

+  -f  +  +  -f  +  +  +  -l-  +  + 

4-4  -t-     + 

Active  Motility. 

«  ?i*f 

+  +  +  +  +  +  +  +  -t-  +  4- 

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Terminal  Flagella. 

=•  S--??1 

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Turbidity. 

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Scum  and  Marked 
Turbidity. 

z£ 
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a  r?X= 

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Very  Slow  Lique- 
faction. 

O 

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QPW(B  j? 

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Slow  Liquefaction. 

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Rapid    Liquefac- 
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Yellowish. 

AGAR- 

£.£*•• 

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000        + 

Grayish. 

AGAR. 

„ 

+            +                4-4- 

+  + 

Yellowish. 

7 

^E 

00              00+             00 

+  00         0 

Brown. 

% 

•°    ^^$- 

o  o          o  o  o          o  o 

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Colorless. 

H 

^°    -    =    'M 

in    '  >~  ~  O 

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n  %  -«  '    3 

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BLOOD- 
SERUM. 

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4_                          _L 

Acidified. 

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S-gs"  o 

•^  c  X       3- 

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Coagulated. 

X 

Ji;":'    X  g 

0000000              0  + 

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Phosphorescent. 

"s^J*^." 
M  -  c/j  ^    •: 

4-             o         + 

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PIGEONS. 

f|?^| 

+        +00        +++++ 

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s-f 

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$r 

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631 

Fluorescent. 

CHAPTER  XXV. 
TYPHOID  FEVER. 

BACILLUS  TYPHOSUS  (EBERTH-GAFFKY). 

General  Characteristics.— A  motile,  flagellated,  non-sporogenous, 
non-liquefying,  nonrchromogenic,  non-aerogenic,  aerobic  and  optionally 
anaerobic,  pathogenic  bacillus,  staining  by  ordinary  methods,  but  not 
by  Gram's  method.  It  does  not  form  indol,  acids  from  sugars,  or  coag- 
ulate milk. 

Typhoid  fever,  "  typhus  abdominalis,"  enteric  fever, 
"la  fievre  typhique,"  is  a  disease  so  well  known  and  of 
such  universal  distribution,  that  no  introductory  remarks 
concerning  it  are  necessary. 

The  bacillus  of  typhoid  fever  (Bacillus  lyphosus)  was 
discovered  in  1880  by  Eberth*  and  Koch,f  and  was  first 
secured  in  pure  culture  from  the  spleen  and  lymphatic 
glands  four  years  later  by  Gaffky.J 


Fig.  220. — Bacillus  typhosus,  from  twenty-four-hour  culture  on 
agar.  (From  Hiss  and  Zinsser,  "Text-Book  of  Bacteriology,"  D.  Ap- 
pleton  &  Co.,  publishers.) 

Distribution. — The  bacillus  is  both  saprophytic  and 
parasitic.  It  finds  abundant  opportunity,  in  nature,  for 
growth  and  development,  and,  enjoying  strong  resisting 
powers,  can  accommodate  itself  to  its  environment  much 
better  than  the  majority  of  pathogenic  bacteria,  and  can 

*  "Virchow's  Archiv,"  1881  and  1883. 

f  "  Mittheilungen  aus  dem  kaiserl.  Gesundheitsamt,"   1,  45, 
id.,  2. 

632 


Morphology — Staining 


633 


be  found  in  water,  air,  soiled  clothing,  dust,  sewage,  milk, 
etc.,  contaminated  directly  or  indirectly  with  the  intestinal 
discharges  of  diseased  persons. 

Morphology. — The  organism  is  a  short,  stout  bacillus, 
about  i  to  3  ft  (2  to  4  ft — Chantemesse,  Widal)  in  length  and 
0.5  to  0.8  U  broad  (Sternberg).  The  ends  are  rounded,  and 
it  is  exceptional  for  the  bacilli  to  be  united  in  chains.  The 
size  and  morphology  vary  with  the  nature  of  the  culture- 
medium  and  the  age  of  the  culture.  Thoinot  and  Masselin,* 
in  describing  these  morphologic  variations,  point  out  that 


Fig.  221. — Bacillus  typhi. 


when  grown  in  bouillon  the  typhoid  bacillus  is  very  slender; 
in  milk  it  is  stouter;  upon  agar-agar  and  potato  it  is  thick 
and  short;  and  in  old  gelatin  cultures  it  forms  long  fila- 
ments. 

Flagella. — The  organisms  are  actively  motile  and  are 
provided  with  numerous  flagella,  which  arise  from  all  parts 
of  the  bacillus  (peritricha) ,  and  are  10  to  20  in  number. 
They  stain  well  by  Loffler's  method.  The  movements  of 
the  short  bacilli  are  oscillating;  those  of  the  longer  bacilli, 
serpentine  and  undulating. 

Staining. — The  organism  stains  quite  well  by  the  ordinary 
methods,  but  loses  the  color  when  stained  by  Gram's  method. 

*  "Precis  de  Microbie,"  Paris,  1893. 


034  Typhoid  Fever 

The  bacillus  gives  up  its  color  in  the  presence  of  almost  any 
solvent,  so  that  it  is  particularly  difficult  to  stain  in  tissue. 

When  sections  of  tissue  containing  the  typhoid  bacilli 
are  to  be  stained,  the  best  method  is  to  allow  them  to  remain 
in  Loffler's  alkaline  methylene-blue  for  from  fifteen  minutes 
to  twenty-four  hours,  then  wash  in  water,  dehydrate  rapidly 
in  alcohol,  clear  up  in  xylol,  and  mount  in  Canada  balsam, 
Ziehl's  method  also  gives  good  results:  The  sections  are 
stained  for  fifteen  minutes  in  a  solution  of  distilled  water 
100,  fuchsin  i,  and  phenol  5.  After  staining  they  are 
washed  in  distilled  water  containing  i  per  cent,  of  acetic 
acid,  dehydrated  in  alcohol,  cleared,  and  mounted.  In  such 
preparations  the  bacilli  are  always  found  in  scattered  groups, 
which  are  easily  discovered,  under  a  low  power  of  the  mi- 
croscope, as  reddish  specks,  and  readily  resolved  into  bacilli 
with  the  oil-immersion  lens. 

In  bacilli  stained  with  the  alkaline  methylene-blue  solu- 
tion, dark-colored  dots  (Babes-Ernst  or  metachromatic 
granules)  may  sometimes  be  observed  near  the  ends  of  the 
rods. 

The  typhoid  bacillus  produces  no  endospores. 

Isolation^ — The  bacillus  can  be  secured  in  pure  culture 
from  an  enlarged  lymphatic  gland  or  from  the  splenic  pulp 
of  a  case  of  typhoid.  To  secure  it  in  this  way  the  autopsy 
should  be  made  as  soon  after  death  as  possible,  lest  the 
colon  bacillus  invade  the  tissues,  and  cause  confusing  con- 
taminations. 

As  the  groups  of  bacilli  are  sometimes  widely  scattered 
throughout  the  spleen,  B.  Frankel  recommends  that  as  soon 
as  the  organ  is  removed  from  the  body  it  be  wrapped  in 
cloths  wet  with  a  solution  of  bichlorid  of  mercury  and  kept 
for  three  days  in  a  warm  room,  in  order  that  a  considerable 
and  massive  development  of  the  bacilli  may  take  place.  The 
surface  is  then  seared  with  a  hot  iron  and  material  for  cultures 
obtained  by  introducing  a  platinum  loop  into  the  substance 
of  the  organ  through  the  sterilized  surface. 

Cultures  of  typhoid  bacillus  may  be  more  easily  obtained 
from  the  blood  of  the  living  patients.  (See  "Blood  culture, * 
under  the  section  "Bacteriologic  Diagnosis.") 

The  bacilli  can  also  be  secured,  but  with  much  less  certainty, 
from  the  alvine  discharges  of  typhoid  patients  during  the 
second  and  third  weeks  of  the  disease. 


Cultivation  635 

Cultivation. — The  bacillus  grows  well  upon  all  culture- 
media  under  both  aerobic  and  anaerobic  conditions. 

Colonies. — The  deep  colonies  upon  gelatin  plates  appear 
under  the  microscope  of  a  brownish-yellow  color  and  spindle  - 
shape,  and  are  sharply  circumscribed.  When  superficial, 
however,  they  become  larger  and  form  a  thin,  bluish,  iri- 
descent layer  with  notched  edges.  The  superficial  colonies 
are  often  described  as  resembling  grapevine  leaves  in  shape. 
The  center  of  the  superficial  colonies  is  the  only  portion 
which  shows  the  yellowish-brown  color.  The.  gelatin  is  not 
liquefied. 

Gelatin  Punctures. — When  transferred  to  gelatin  punc- 
ture cultures,  the  typhoid  bacilli  develop  along  the  entire 


Fig.    222. — Bacillus   typhi   abdominalis;    superficial    colony  two   days 
old,  as  seen  upon  the  surface  of  a  gelatin  plate.      X  20  (Heim). 

track  of  the  wire,  with  the  formation  of  minute,  confluent, 
spheric  colonies.  A  small,  thin,  whitish  layer  develops 
upon  the  surface  near  the  center.  The  gelatin  is  not  lique- 
fied, but  is  sometimes  slightly  clouded  in  the  neighborhood 
of  the  growth. 

Agar-agar. — The  growth  upon  the  surface  of  obliquely 
solidified  gelatin,  agar-agar,  or  blood-serum  is  not  luxuriant. 
It  forms  a  thin,  moist,  shining,  translucent  band  with  smooth 
edges  and  a  grayish-yellow  color. 

Potato. — When  a  potato  is  inoculated  and  stood  in  the 
incubating  oven,  the  typical  growth  cannot  be  detected 
even  at  the  end  of  the  second  day,  unless  the  observer  be 
skilled  and  the  examination  thorough.  If,  however,  the 


636  Typhoid  Fever 

surface  of  the  medium  be  touched  with  a  platinum  wire,  it 
is  found  that  its  entire  surface  is  covered  with  a  rather 
thick,  invisible  layer  of  a  sticky  vegetation  which  the  mi- 
croscope shows  to  be  made  up  of  bacilli.  This  is  described 
as  the  invisible  growth.  Unfortunately,  it  is  not  a  constant 
characteristic,  for  occasionally  a  typhoid  bacillus  will  show 
a  distinct  yellowish  or  brownish  color.  The  typical  growth 
seems  to  take  place  only  when  the  reaction  of  the  potato 
is  acid. 

Bouillon. — Jn  bouillon  the  only  change  produced  by 
the  growth  of  the  bacillus  is  a  diffuse  cloudiness.  Rarely  a 
pellicle  is  formed.  When  sugars  are  added  to  the  bouillon 
the  typhoid  bacillus  is  found  to  form  acid  from  dextrose, 
levulose,  galactose,  mannite,  maltose,  and  dextrin,  but  not  to 
form  acid  from  lactose  or  saccharose.  No  gas  is  formed  in 
the  fermentation  tube  with  any  of  the  sugars. 

Milk. — In  milk  containing  litmus  a  very  slight  and  slow 
acidity  is  produced,  which  later  gives  rise  to  a  distinct  alka- 
linity. The  milk  is  not  coagulated. 

Vital  Resistance. — The  organisms  grow  well  at  all 
ordinary  temperatures.  The  thermal  death-point  is  given 
by  Sternberg  as  56°  C.,  destruction  being  effected  in  ten 
minutes.  Upon  ordinary  culture-media,  the  organisms  re- 
main alive  for  several  months  if  drying  is  prevented.  In 
carefully  sealed  agar-agar  tubes  Hiss  found  the  organism 
still  living  after  thirteen  years.  According  to  Klemperer  and 
Levy,*  the  bacilli  can  remain  vital  for  three  months  in  dis- 
tilled water,  though  in  ordinary  water  the  commoner  and 
more  vigorous  saprophytes  outgrow  them  and  cause  their  dis- 
appearance in  a  few  days.  There  seems  to  be  some  doubt, 
however,  on  this  point,  as  Tavelf  found  that  it  lived  for 
six  months  in  the  blind  terminal  of  a  water-supply  pipe, 
and  Hofmann,  {  after  planting  it  in  an  aquarium  containing 
fish,  snails,  water-plants,  and  protozoa,  was  able  to  recover 
it  from  the  water  after  thirty-six  days,  and  from  the  mud 
in  the  bottom  after  two  months.  In  elaborate  experimental 
studies  of  this  question  Jordan,  Russell,  and  Zeit§  found 
its  longevity  to  be  only  three  or  four  days  under  conditions 

*"  Clinical  Bacteriology."  Translated  by  A.  A.  Eshner,  Phila.j 
W.  B.  Saunders  Co.,  1900. 

t  "Centralbl.  f.  Bakt.  u,  Parasitenk.,"  xxxm,  p.  166,  1903. 

t  "Archiv.  f.  Hyg.,"  1905,  LII,  2,  208. 

§  "Journal  of  Infectious  Diseases,"  1904,  i,  p.  641. 


Toxic  Products  637 

simulating  as  nearly  as  possible  those  found  in  nature 
When  buried  in  the  upper  layers  of  the  soil  the  bacilli 
retain  their  vitality  for  nearly  six  months.  Robertson* 
found  that  when  planted  in  soil  and  occasionally  fed 
by  pouring  bouillon  upon  the  surface,  the  typhoid  bacillus 
maintained  its  vitality  for  twelve  months.  He  suggests 
that  it  may  do  the  same  in  the  soil  about  leaky  drains. 

Cold  has  little  effect  upon  typhoid  bacilli,  for  some  can 
withstand  freezing  and  thawing  several  times.  Observing 
that  epidemics  of  typhoid  fever  had  never  been  traced  to 
polluted  ice,  Sedgwick  and  Winslowf  made  some  investiga- 
tions to  determine  what  quantitative  reduction  might  be 
brought  about  by  freezing,  and  accordingly  experimentally 
froze  a  large  number  of  samples  of  water  intentionally  in- 
fected with  large  numbers  of  typhoid  bacilli  from  different 
sources.  It  was  found  that  the  typhoid  bacilli  disappeared 
in  proportion  to  the  length  of  time  the  water  was  frozen, 
and  that  the  reduction  averaged  99  per  cent,  in  two  weeks. 
The  last  two  or  three  germs  per  thousand  appeared  very 
resistant  and  sometimes  remained  alive  after  twelve  weeks. 

They  have  been  found  to  remain  alive  upon  linen  from 
sixty  to  seventy-two  days,  and  upon  buckskin  from  eighty 
to  eighty-five  days. 

The  typhoid  bacillus  resists  the  action  of  chemic  agents 
rather  better  than  most  non-sporogenous  organisms.  The 
addition  of  from  o.i  to  0.2  per  cent,  of  carbolic  acid  to  the 
culture-media  being  without  effect  upon  its  growth.  At 
one  time  the  tolerance  to  carbolic  acid  was  thought  to  be 
characteristic,  but  it  is  now  known  to  be  shared  by  other 
bacteria  (colon  bacillus) .  It  is  killed  by  i  :  500  bichlorid  of 
mercury  solutions  and  5  per  cent,  carbolic  acid  solutions  in 
five  minutes. 

Metabolic  Products. — The  typhoid  bacillus  does  not 
produce  indol.  It  produces  a  small  amount  of  acid  when 
grown  in  sugar-containing  media,  but  its  regular  tendency  is  to 
form  alkalies,  as  is  shown  by  the  reactions  in  litmus  milk. 
It  forms  no  coagulating  or  proteolytic  enzymes. 

Toxic  Products. — The  disproportion  of  local  to  consti- 
tutional disturbance  in  typhoid  fever  and  the  irritative 
and  necrotic  character  of  its  lesions  suggest  that  we  have 

*  "Brit.  Med.  Jour.,"  Jan.  8,  1898. 

f"Jour.  Boston  Soc.  of  Med.  Sci.,"  vol.  iv,  No.  7,  p.  181,  March 
20,  1900. 


638  Typhoid  Fever 

to  do  with  a  toxic  organism.  Brieger  and  Frankel  have, 
indeed,  separated  a  toxalbumin,  which  they  thought  to  be 
the  specific  poison,  from  bouillon  cultures.  When  injected 
into  guinea-pigs  the  typhotoxin  of  Brieger  'causes  salivation, 
accelerated  respiration,  diarrhea,  mydriasis,  and  death  in 
from  twenty-four  to  forty-eight  hours.  Klemperer  and  Levy 
also  point  out,  as  affording  clinical  proof  of  the  presence  of 
toxin,  the  occasional  fatal  cases  in  which  the  typical  picture 
of  typhoid  has  been  without  the  characteristic  postmortem 
lesions,  the  diagnosis  being  made  by  the  discovery  of  the 
bacilli  in  the  spleen. 

Pfeiffer  and  Kolle*  found  toxic  substance  in  the  bodies 
of  the  bacilli  only.  It  was  not,  like  the  toxins  of  diph- 
theria and  tetanus,  dissolved  in  the  culture -medium.  This 
was  an  obstacle  to  the  immunization  experiments  of  both 
Pfeiffer  and  Kolle  and  Lofner  and  Abel,  f  for  the  only  method 
of  immunizing  animals  was  to  make  massive  agar-agar 
cultures,  scrape  the  bacilli  from  the  surface,  and  distribute 
them  through  an  indifferent  fluid  before  injecting  them  into 
animals. 

If  the  bacilli  grown  upon  ordinary  culture-media  are 
several  times  washed  in  distilled  water,  and  then  allowed 
to  macerate  in  normal  salt  solution,  autolysis  takes  place 
and  a  toxin  is  liberated,  showing  that  the  toxin  is  intracel- 
lular.  Macfadyen  and  Rowland  t  liberated  an  intracellular 
toxin  from  cultures  of  the  typhoid  bacilli  by  freezing  them 
with  liquid  air  and  grinding  them  in  an  agate  mortar.  Ani- 
mals immunized  with  this  poison  produced  an  antiserum 
active  against  it,  but  useless  against  infection  with  typhoid 
bacilli.  Wright,  of  Netley,  §  observes  that  Macfadyen's 
method  of  securing  this  intracellular  toxin  was  unneces- 
sarily cumbersome,  as  the  body  juices  of  animals  injected 
with  dead  cultures  of  the  bacilli  dissolve  them  at  once  and 
thus  liberate  the  same  toxic  product. 

Besredka||  and  Macfadyen**  think  that  exotoxin  is  also 
forrned.  Vaughanff  has  obtained  poisonous  and  non- 

*  "Deutsche  med.  Wochenschrift,"  Nov.  12,  1896. 

t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Jan.  23,  1896,  Bd.  xix,  No. 
23,  P-  Si- 

J  "Brit.  Med.  Jour.,"  1903. 

§  Ibid.,  April,  4  1903,  i,  p.  786. 

||  "Ann.  de  1'Inst.  Pasteur,"  1895,  x,  1896,  xi. 
**  "Centralbl.  f.  Bakt.,"  etc.,  1906,  i. 
ft  "Amer.  Jour.  Med.  Sci.,"  1908,  cxxxvi. 


Pathogenesis  639 

poisonous  fractions  by  extracting  massive  cultures  of  typhoid 
bacilli  with  2  per  cent,  solutions  of  sodium  hydrate  in  abso- 
lute alcohol  at  78°  C. 

Mode  of  Infection. — The  typhoid  bacillus  enters  the  body 
by  way  of  the  alimentary  tract  with  infected  foods  and  drinks. 

Rosenau,  Lumsden,  and  Kastle*  were  able  to  connect  10 
per  cent,  of  the  cases  occurring  in  the  District  of  Columbia 
with  infection  through  milk.  Interesting  additional  facts 
upon  the  subject  can  be  found  in  Bulletin  No.  41  of  the 
Hygienic  Laboratory  upon  "Milk  in  its  Relation  to  the 
Public  Health."  The  bacillus  occasionally  enters  milk  in 
water  used  to  dilute  it  or  to  wash  the  cans. 

The  occurrence  of  typhoid  fever  among  the  soldiers  of  the 
United  States  Army  during  the  Spanish-American  War  in 
1898  was  shown  by  Reed,  Vaughan,  and  Shakespeare  f  to 
be  largely  the  result  of  the  pollution  of  the  food  of  the  soldiers 
by  flies  that  shortly  before  had  visited  infected  latrines. 

The  bacillus  is  also  occasionally  present  upon  green  vege- 
tables grown  in  soil  fertilized  with  infected  human  excre- 
ment or  sprinkled  with  polluted  water,  and  epidemics  are 
reported  in  which  the  occurrence  of  the  disease  was  traced 
to  oysters  infected  through  sewage.  NewsholmeJ  found 
that  in  56  cases  of  typhoid  fever  about  one-third  were  at- 
tributable to  eating  raw  shell-fish  from  sewage-polluted  beds. 

Pathogenesis. — The  primary  activities  of  the  typhoid 
bacillus  are  unknown.  It  is  supposed  that  it  passes  uninjured 
through  the  acid  secretions  of  the  stomach  to  enter  the  intes- 
tine, where  local  disturbances  are  set  up.  Whether  dur- 
ing an  early  residence  in  the  intestine  its  metabolism  is 
accompanied  by  the  formation  of  a  toxic  product,  irritating 
to  the  mucosa,  and  affording  the  bacilli  means  of  entrance 
to  the  lymph-vessels,  through  diminutive  breaches  of  con- 
tinuity, is  not  known.  We  usually  find  it  well  established 
in  the  intestinal  and  mesenteric  lymphatics  at  the  time  we 
are  able  to  recognize  the  disease,  though  in  rare  cases  it 
appears  able  to  reach  the  blood  through  other  than  the 
customary  channels  and  occasion  an  entirely  different 
pathologic  picture. 

It  is  quite  certain  that  the  chief  operations  of  the  typhoid 

*  "Hygienic  Laboratory  Bulletin  No.  33,"  Washington,  D.  C.,  1907. 
t  "Report  on  Typhoid  Fever  in  the  U.  S.  Military  Camps  in  the  Span- 
ish War,"  vol.  I. 

J"Brit.  Med.  Jour.,"  Jan.  1895. 


640  Typhoid  Fever 

bacillus  are  in  the  tissues  and  not  in  the  intestine,  as  seems 
to  be  a  widely  prevalent  error.  It  is  contrary  to  most  of 
our  knowledge  of  the  organism  that  it  should  easily  adapt 
itself  to  saprophytic  existence  among  the  more  vigorous 
intestinal  organisms.  Those  who  look  for  it  in  the  feces 
are  usually  surprised  at  the  difficulty  of  finding  it,  or  at  the 
small  numbers  they  succeed  in  isolating.  It  is  far  more 
easy  to  isolate  the  organism  from  the  blood  than  from  the 
feces,  and  much  greater  numbers  occur  in  the  urine  than  in 
the  feces.  It  probably  escapes  from  the  blood  into  the 
bile,  where  it  grows  luxuriantly,  and  entering  the  gall-bladder 
may  take  up  permanent  residence  there,  escaping  into  the 
intestine  each  time  the  gall-bladder  is  emptied.  Many 
bacilli  thus  discharged  probably  meet  with  destruction  in  the 
intestine,  though  some  convalescents  from  typhoid  fever  for 
years  have  a  periodic  appearance  of  bacilli  in  the  feces. 
Such  individuals  have  become  known  as  "typhoid  carriers" 
and  are  a  menace  to  the  public. 

In  a  case  studied  by  Miller*  they  were  found  in  the  gall- 
bladder seven  years  after  recovery  from  typhoid  fever;  in  a 
case  studied  by  Drobaf  bacilli  were  found  in  both  the  gall- 
bladder and  a  gall-stone  seventeen  years  after  recovery  from 
typhoid  fever ;  in  a  case  of  Humer,  J  in  the  gall-bladder  of  a 
patient  suffering  from  cholecystitis,  eighteen  years  after  re- 
covery from  an  attack  of  typhoid  fever,  and  in  a  case  studied 
by  Dean,§'  in  the  stools  of  a  man  twenty-nine  years  after 
he  had  had  typhoid  fever,  and  who  had  probably  been 
carrying  them  about  in  his  gall-bladder  ever  since.  Gush- 
ing ||  invariably  found  the  bacilli  in  the  bile  in  clumps  re- 
sembling the  agglutinations  of  the  Widal  reaction.  He 
thinks  it  probable  that  these  clumps  form  nuclei  upon  which 
bile  salts  can  be  precipitated  and  calculous  formation  begun. 
The  presence  of  gall-stones,  together  with  the  long-lived 
infective  agents,  may  at  any  subsequent  time  provoke  a 
cholecystitis.  Gushing  collected  6  cases  of  operation  for 
cholecystitis  with  calculi  in  which  typhoid  bacilli  were 
present,  and  5  in  which  Bacillus  coli  communis  was  present 
in  the  gall-bladder. 

*  "Bull,  of  the  Johns  Hopkins  Hospital,"  May,i898. 

t  "Wiener  klin.  Wochenschrift,"  1899,  xn,  p.  1141. 

J"Bull.  of  the  Johns  Hopkins  Hospital,"  Aug.  and  Sept.,   1899. 

§  "British  Medical  Journal,"  March  7,  1908,  I,  p.  562. 

||  "Bull,  of  the  Johns  Hopkins  Hospital,"  ix,  No.  86. 


Pathogenesis 


641 


With  the  most  approved  methods  yet  devised,  Peabody 
and  Pratt*  were  unable  to  recover  the  micro-organism  from 
the  intestinal  contents  in  more  than  21  per  cent,  of  febrile 
cases,  and  only  in  small  numbers  as  a  rule.  The  greatest 
number  was  obtained  when  there  was  much  blood  in  the 
stool. 

There  is  always  well-marked  blood-infection  during  the 
first  couple  of  weeks  of  the  disease,  and  upon  this  depends 
the  occurrence  of  the  rose-colored  spots. 

The  bacilli  enter  the  solitary  glands  and  Peyer's  patches, 
and  multiply  slowly  during  the  incubation  period  of  the 


Fig.  223. — Intestinal  perforation  in  typhoid  fever.  Observe  the 
threads  of  tissue  obstructing  the  opening.  (Museum  of  the  Penn- 
sylvania Hospital.)  (Keen,  "Surgical  Complications  and  Sequels  of 
Typhoid  Fever.") 

disease — one  to  three  weeks.  The  immediate  result  of  their 
activity  in  the  lymphatic  structures  is  an  increase  in  the 
number  of  cells,  and  ultimately  necrosis  and  sloughing  of 
the  Peyer's  patches  and  solitary  glands  (Fig.  223).  From 
the  intestinal  lymphatics  the  bacilli  pass,  in  all  probability, 

*  "Journal  of  the  American  Medical   Association,"   Sept.   7,    1907, 
xux,  p.  846. 
41 


642  Typhoid  Fever 

to  the  mesenteric  glands,  which  become  enlarged,  softened, 
and  sometimes  rupture.  They  also  invade  the  spleen,  liver, 
and  sometimes  the  kidneys,  and  other  organs  where  they 
may  be  found  always  aggregated  in  small  clusters  in  properly 
stained  specimens.  The  occurrence  of  the  bacilli  in  the 
tissues  in  clumps  or  clusters  may  depend  upon  the  presence 
of  agglutinating  substances  in  the  blood. 

Mallory  *  found  the  histologic  lesions  of  typhoid  fever  to 
be  widespread  throughout  the  body  and  not  limited  to  the 
Peyer's  patches  of  the  intestine,  where  they  are  most 
evident.  His  conclusions  regarding  the  pathogenesis  of  the 
disease  are  briefly:  "The  typhoid  bacillus  produces  a  mild 
diffusible  toxin,  partly  within  the  intestinal  tract,  partly 
within  the  blood  and  organs  of  the  body.  This  toxin  pro- 
duces proliferation  of  the  endothelial  cells,  which  acquire 
for  a  certain  length  of  time  malignant  properties.  The  new- 
formed  cells  are  epithelioid  in  character,  have  irregular, 
lightly  staining,  eccentrically  situated  nuclei,  abundant, 
sharply  defined,  acidophilic  protoplasm,  and  are  charac- 
terized by  marked  phagocytic  properties.  These  phago- 
cytic  cells  are  produced  most  abundantly  along  the  line  of 
absorption  from  the  intestinal  tract,  both  in  the  lymphatic 
apparatus  and  in  the  blood-vessels.  They  are  also  produced 
by  distribution  of  the  toxin  through  the  general  circulation, 
in  greatest  numbers  where  the  circulation  is  slowest.  Finally, 
they  are  produced  all  over  the  body  in  the  lymphatic  spaces 
and  vessels  by  absorption  of  the  toxin  eliminated  from  the 
blood-vessels.  The  swelling  of  the  intestinal  lymphoid  tissue 
of  the  mesenteric  lymph-nodes  and  of  the  spleen  is  due 
almost  entirely  to  the  formation  of  phagocytic  cells.  The 
necrosis  of  the  intestinal  lymphoid  tissue  is  accidental  in 
nature  and  is  caused  through  occlusion  of  the  veins  and 
capillaries  by  fibrinous  thrombi,  which  owe  their  origin  to 
degeneration  of  phagocytic  cells  beneath  the  lining  endo- 
thelium  of  the  vessels.  Two  varieties  of  focal  lesions  occur 
in  the  liver :  one  consists  of  the  formation  of  phagocytic  cells 
in  the  lymph-spaces  and  vessels  around  the  portal  vessels 
under  the  action  of  the  toxin  absorbed  by  the  lymphatics; 
the  other  is  due  to  obstruction  of  liver  capillaries  by  phago- 
cytic cells  derived  in  small  part  from  the  lining  endothe- 
lium  of  the  liver  capillaries,  but  chiefly  by  embolism  through 
the  portal  circulation  of  cells  originating  from  the  endo- 
*  "Journal  of  Experimental  Medicine,"  vol.  m,  1898,  p.  611. 


Pathogenesis  643 

thelium  of  the  blood-vessels  of  the  intestine  and  spleen. 
The  liver-cells  lying  between  the  occluded  capillaries  undergo 
necrosis  and  disappear.  Later  the  foci  of  cells  degenerate 
and  fibrin  forms  between  them.  Invasion  by  polymorpho- 
nuclear  leukocytes  is  rare." 

".  .  .  Histologically  the  typhoid  process  is  prolifera- 
tive  and  stands  in  close  relationship  to  tuberculosis,  but  the 
lesions  are  diffuse  and  bear  no  intimate  relation  to  the 
typhoid  bacillus,  while  the  tubercular  process  is  focal  and 
stands  in  the  closest  relation  to  the  tubercle  bacillus." 

The  growth  of  the  bacilli  in  the  kidneys  causes  albu- 
minuria,  and  the  bacilli  can  be  found  in  the  urine  in  about 
25  per  cent,  of  the  cases.  Smith  *  found  them  in  the 
urine  in  3  out  of  7  cases  which  he  investigated;  Richardson, f 
in  9  out  of  38  cases.  They  did  not  occur  before  the  third 
week,  and  remained  in  one  case  twenty-two  days  after 
cessation  of  the  fever.  Sometimes  they  were  present  in 
immense  numbers,  the  urine  being  actually  clouded  by  their 
presence.  Petruschky  J  found  that  albuminuria  sometimes 
occurs  without  the  presence  of  the  bacilli;  that  their 
presence  in  the  urine  is  infrequent;  that  the  bacilli  never 
appear  in  the  urine  in  the  early  part  of  the  disease,  and 
hence  are  of  little  importance  for  diagnostic  purposes. 
Gwyn  §  has  found  as  many  as  50,000,000  typhoid  bacilli 
per  cubic  centimeter  of  urine,  and  mentions  a  case  of  Gush- 
ing's  in  which  the  bacilli  persisted  in  the  urine  jor  six  years 
after  the  primary  attack  of  typhoid  fever.  Their  occurrence, 
no  doubt,  depends  primarily  upon  a  typhoid  bacteremia, 
by  which  they  are  brought  to  the  kidney.  After  recovery 
from  typhoid  fever,  their  persistence  in  the  urine  depends 
upon  continued  growth  in  the  bladder  and  not  upon  con- 
tinuous escape  from  the  blood.  It  is  of  importance  from 
a  sanitary  point  of  view  to  remember  that  the  urine  as  well 
as  the  feces  is  infectious. 

The  bacilli  pass  from  the  lymphatics  to  the  general 
circulation,  so  that  all  cases  of  typhoid  fever  are  true 
bacteremias  during  part  or  all  of  their  course. 

Ordinarily  few  bacilli  can  be  found  in  the  circulatory 
blood,  but  blood  from  the  roseolas  contains  them,  and  the 

*  "Brit.  Med.  Jour.,"  Feb.  13,  1897. 

f  "Journal  of  Experimental  Medicine,"  May,  1898. 

%  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  May  13,  1898,  No.  13,  p.  577, 

\  "Phila.  Med.  Jour.,"  March  3,  1900. 


644  Typhoid  Fever 

eruption  may  be  regarded  as  one  of  the  local  irritative 
manifestations  of  the  bacillus. 

Particularly  careful  work  upon  this  subject  has  been 
done  by  Richardson,*  who  found  that  by  carefully  disin- 
fecting the  skin,  freezing  it  with  chlorid  of  ethyl,  making 
a  crucial  incision,  and  cultivating  from  the  blood  thus 
obtained,  he  was  able  to  secure  the  typhoid  bacillus  in 
13  out  of  14  cases  examined.  It  was,  however,  necessary  to 
examine  a  number  of  spots  in  each  case. 

As  a  means  of  diagnosis  the  matter  is  of  some  importance, 
as  the  rose  spots  may  precede  the  occurrence  of  the  Widal 
reaction  by  a  number  of  days. 

In  rare  instances  the  bacillus  may  be  found  in  the  expec- 
toration, especially  when  pulmonary  complications  arise  in 
the  course  of  the  disease.  Cases  of  this  kind  have  been  re- 
ported by  Chantemesse  and  Widal  f  and  Frankel.f 

The  pyogenic  power  of  the  typhoid  bacillus  was  first 
pointed  out  by  A.  Frankel,  who  observed  it  in  a  suppuration 
that  occurred  four  months  after  convalescence.  Low§  found 
virulent  typhoid  bacilli  in  the  pus  of  abscesses  occurring 
from  one  to  six  years  after  convalescence. 

Weichselbaum  has  seen  general  peritonitis  from  rupture 
of  the  spleen  in  typhoid  fever  with  escape  of  the  bacilli. 
Otitis  media,  ostitis,  periostitis,  and  osteomyelitis  are  very 
common  results  of  the  lodgment  of  the  bacilli  in  bony  tis- 
sue. Ohlmacherll  has  found  the  bacilli  in  suppurations  of 
the  membranes  of  the  brain.  The  bacilli  are  also  encoun- 
tered in  other  local  suppurations  occurring  in  or  following 
typhoid  fever.  Flexner  and  Harris**  have  seen  a  case  in 
which  the  distribution  of  the  bacilli  was  sufficiently  wide- 
spread to  constitute  a  real  septicemia,  the  bacillus  being 
isolated  from  various  organs  of  the  body. 

Lower  Animals. — Typhoid  fever  is  communicable  to 
animals  with  difficulty.  They  are  not  infected  by  bacilli 
contained  in  fecal  matter  or  by  the  pure  cultures  mixed  with 
the  food,  and  are  not  injured  by  the  injection  of  blood  from 
typhoid  patients.  Gaffky  failed  completely  to  produce  any 

"Phila.  Med.  Jour.,"  March  3,  1900. 
t  "Archiv.  de  physiol.  norm.  et.  path.,"  1887. 
J  "Deutsche  med.  Wochenschrift,"  1899,  xv,  xvi. 
§  "Sitz.  der  k.  k.  Gesellschaft  d.  Aerzt.  in  Wien,"  "Aerztl.  Central- 
Anz.,"  1898,  No.  3. 

||  "Jour.  Amer.  Med.  Assoc.,"  Aug.  28,  1897. 
**  "Bull.  Johns  Hopkins  Hospital,"  Dec.,  1897. 


Prophylaxis  645 

symptoms  suggestive  of  typhoid  fever  in  rabbits,  guinea- 
pigs,  white  rats,  mice,  pigeons,  chickens,  and  calves,  and 
found  that  Java  apes  could  feed  daily  upon  food  polluted 
with  typhoid  bacilli  for  a  considerable  time,  yet  without 
symptoms.  Grunbaum*  produced  typhoid  fever  in  the 
chimpanzee  by  inoculation  with  the  bacillus.  This  seems 
to  prove  its  specific  nature.  The  introduction  of  virulent 
cultures  into  the  abdominal  cavity  of  animals  is  followed  by 
peritonitis. 

Germano  and  Maureaf  found  that  mice  succumbed  in 
from  one  to  three  days  after  intraperitoneal  injection  of 
i  or  2  c.c.  of  a  twenty-four-hour-old  bouillon  culture.  Sub- 
cutaneous injections  in  rabbits  and  dogs  caused  abscesses. 

Losener  found  the  introduction  of  3  mg.  of  an  agar-agar 
culture  into  the  abdominal  cavity  of  guinea-pigs  to  be 
fatal. 

Petruschky  t  found  that  mice  convalescent  from  sub- 
cutaneous injections  of  typhoid  cultures  frequently  suffered 
from  a  more  or  less  widespread  necrosis  of  the  skin  at  the 
point  of  injection. 

Prophylaxis. — One  of  the  most  important  and  practical 
points  for  the  physician  to  grasp  in  relation  to  the  subject  of 
typhoid  fever  is  the  highly  virulent  character  of  the  dis- 
charges, both  feces  and  urine.  In  every  case  the  greatest 
care  should  be  taken  for  their  proper  disinfection,  a  rigid 
attention  paid  to  all  the  details  of  cleanliness  in  the  sick- 
room, and  the  careful  sterilization  of  all  articles  which  are 
soiled  by  the  patient.  If  country  practitioners  were  as 
careful  in  this  particular  as  they  should  be,  the  disease 
\\ould  be  much  less  frequent  in  regions  remote  from  the 
filth  and  squalor  of  large  cities  with  their  unmanageable 
slums,  and  the  distribution  of  the  bacilli  to  villages  and 
towns,  by  milk  and  by  watercourses  polluted  in  their  in- 
fancy, might  be  checked. 

In  large  cities  where  typhoid  fever  has  been  endemic  the 
incidence  of  the  disease  has  been  enormously  reduced  by 
purification  of  the  water-supply.  Where  this  measure  is  not 
possible,  the  safety  of  the  individual  citizens  can  be  promoted 
by  using  bottled  pure  waters  for  drinking  purposes  or  by 
boiling  the  water  for  domestic  consumption. 

*  "Brit.  Med.  Jour.,"  April  9,  1904. 

f  "Ziegler's  Beitrage,"  Bd.  xn,  Heft,  3,  p.  494. 

%  "Zeitschrift  fur  Hygiene,"  1892,  Bd.  xn,  p.  261. 


646  Typhoid  Fever 

In  military  camps,  etc.,  the  fly  as  a  carrier  of  the  infection 
must  first  be  excluded  from  the  latrines  and  then  as  well  from 
the  kitchens  and  mess  tents.  When  epidemics  are  in  prog- 
ress, green  vegetables  and  oysters  that  may  be  polluted  by 
infected  water  must  be  guarded  against. 

Prophylactic  Vaccination. — Following  the  principle  of 
Haffkine's  anticholera  inoculations,  Pfeiffer  and  Kolle,* 
Wright,!  and  Wright  and  SempleJ  have  used  subcutaneous 
injections  of  sterilized  cultures  as  a  prophylactic  measure. 
One  cubic  centimeter  of  a  bouillon  culture  sterilized  by  heat 
was  used. 

The  "Indian  Medical  Gazette"  gives  the  following  im- 
portant figures  showing  what  was  accomplished  in  1899: 
Among  the  British  troops  in  India  there  were  1312  cases  of 
typhoid  fever,  with  348  deaths  (25  per  cent.).  The  ratio  of 
admissions  to  the  total  strength  was  20.6  per  1000.  There 
were  4502  inoculations,  and  among  them  there  were  only  9 
deaths  from  typhoid  fever — 0.2  per  cent,  of  the  strength. 
There  were  44  admissions,  giving  0.98  per  cent,  of  the 
strength.  Among  the  non-inoculated  men  of  the  same 
corps  and  at  the  same  stations,  of  25,851  men  there  were 
657  cases  and  146  deaths,  giving  the  relative  percentages 
of  admissions  and  deaths  as  2.54  and  0.56. § 

In  a  later  contribution,  Wright  ||  showed  that  the  pro- 
phylactic vaccination  against  typhoid  fever  reduced  the 
number  of  cases  and  diminished  the  death-rate  among  the 
inoculated,  and  also  called  attention  to  the  slight  risk  the 
inoculated  run  of  being  injured  in  case  their  vital  resistance 
is  below  normal,  or  they  are  already  in  the  early  stages  of 
the  disease,  or  where  the  dose  administered  is  too  large  or 
the  second  vaccination  given  too  soon  after  the  first. 

In  1903  Wright**  published  new  statistics  on  the  subject, 
and  between  1903  and  1908  numerous  references  to  the 
subject  appear  in  the  "British  Medical  Journal,"  in  the 
"Lancet,"  and  in  the  "Journal  of  the  Royal  Army  Medical 
Corps,"  all  favorable  in  their  general  attitude. 

During  the  Mexican  Revolution  of  1911,  the  United  States 

*"  Deutsche  med.  Wochenschrift,"  1896,  xxn;  1898,  xxiv. 
t  "Lancet,"  Sept.,  1896. 
J  "Brit.  Med.  Jour.,"  1897,  i,  p.  256. 
§  "Phila.  Med.  Jour.,"  Oct.  13,  1900,  p.  688. 
||  "The  Lancet,"  Sept.  6,  1902. 
**  "Brit.  Med.  Jour.,"  Oct.  10,  1903. 


Specific  Therapy  647 

Government  began,  on  March  10,  1911,  the  mobilization  of 
regiments  of  the  United  States  Army  on  the  Mexican  fron- 
tier near  San  Antonio,  Texas.  In  order  to  prevent  the  sad 
experiences  of  the  Spanish-American  War,  in  which  the 
troops  suffered  terribly  from  typhoid  fever,  the  Secretary 
of  War  determined  that  the  entire  command  should  be  im- 
munized against  the  disease.  Many  of  the  soldiers  arriving 
on  the  ground  had  already  been  immunized,  the  remainder 
were  at  once  given  the  necessary  injections  upon  arrival. 
The  mean  strength  of  the  command  at  San  Antonio  was  12,000 
up  to  June  30,  1911,  a  period  approximating  four  months. 
During  all  that  time  there  were  only  2  cases  of  typhoid  fever 
in  the  encampment,  i  in  an  uninoculated  civilian  teamster 
and  i  in  an  inoculated  soldier.  Both  cases  recovered.  The 
soldier  suffered  from  so  mild  an  attack  that  it  was  over  in 
sixteen  days  and  probably  would  not  have  been  diagnosed  had 
not  a  blood-culture  been  made.  During  the  four  months 
from  March  loth  to  June  3oth  the  typhoid  fever  was 
prevalent  in  San  Antonio,  there  being  49  cases  with  19 
deaths.* 

The  prophylactic  used  was  prepared  from  a  specially  se- 
lected strain  of  Bacillus  typhosis  grown  on  agar-agar  in  Kolle 
flasks  for  twenty-four  hours.  The  growth  was  washed  off 
with  normal  salt  solution,  killed  by  heating  at  55°  to  56°  C.  in 
a  water-bath,  standardized  by  counting  the  bacteria  accord- 
ing to  the  method  of  Wright,  and  after  being  diluted  with  salt 
solution,  0.25  per  cent,  of  trikresol  was  added.  One  cubic  cen- 
timeter of  the  finished  prophylactic  contained  i  ,000,000,000 
bacilli.  The  first  dose  injected  contained  500,000,000  bacilli, 
the  second  and  third,  given  after  ten  and  twenty  days,  con- 
tained 1,000,000,000  each.  The  injections  caused  little  in- 
convenience either  locally  or  constitutionally.  Only  i  case 
had  fever,  chills,  and  sweats,  and  this  was  the  only  case  requir- 
ing treatment  in  the  hospital.  It  subsequently  developed 
that  this  soldier  was  suffering  from  early  tuberculosis, 
which  may  explain  the  untoward  symptoms  from  which  he 
suffered. 

Specific  Therapy. — Animals  can  be  immunized  to  this 
bacillus,  and  then,  according  to  Chantemesse  and  Widal,  de- 
velop antitoxic  blood  capable  of  protecting  other  animals. 

*  ''Report  of  the  Surgeon-General  of  the  United  States  Army  to  the 
Secretary  of  War,"  1911,  Washington,  D.  C. 


648  Typhoid  Fever 

Stern*  found  in  the  blood  of  human  convalescents  a  substance 
thought  to  have  a  protective  effect  upon  infected  guinea-pigs. 
His  observation  is  in  accordance  with  a  previous  one  by 
Chantemesse  and  Widal,  and  has  recently  been  abundantly 
confirmed. 

The  immunization  of  dogs  and  goats  by  the  introduction 
of  increasing  doses  of  virulent  cultures  has  been  achieved 
by  Pfeiffer  and  Kolle  f  and  by  Loffler  and  Abel.  |  From  these 
animals  immune  serums  were  secured. 

Walger§  reported  4  cases  treated  successfully  with  a  serum 
obtained  from  convalescent  patients.  Ten  cubic  centimeters 
were  given  at  a  dose,  and  the  injection  was  repeated  in  i  case 
with  relapse. 

Rumpfll  and  Kraus  and  Buswell**  report  a  number  of 
cases  of  typhoid  favorably  influenced  by  hypodermic  injec- 
tions of  small  doses  of  sterilized  cultures  of  Bacillus  pyo- 
cyaneus. 

Jez  ft  believes  that  the  antitoxic  principle  in  typhoid  fever 
is  contained  in  some  of  the  internal  organs  instead  of  the 
blood,  and  claims  to  have  obtained  remarkable  results  in 
1 8  cases  treated  with  extracts  of  the  bone-marrow,  spleen, 
and  thymus  of  rabbits  previously  injected  with  the  typhoid 
bacillus. 

Chantemesse,  it  Pope,§§  and  Steele||  ||  have  all  used  serums 
from  animals  immunized  against  typhoid  cultures  for  the 
treatment  of  typhoid  fever,  with  more  or  less  success.  An 
analysis  of  the  results  will,  however,  show  them  to  be  very 
inconclusive. 

The  serum  prepared  by  Macfadyen,***  by  crushing  cul- 
tures frozen  with  liquid  air  and  injecting  animals  with  the 
thus  liberated  intracellular  toxin,  seems  to  be  no  improve- 
ment upon  others. 

*  " Zeitschrif t  fur  Hygiene,"  1894,  xvi,  p.  458. 

t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Jan.,  23,  1896,  Bd.  xix,  No. 
23,  P-  51- 

J  Ibid,  1896. 

§  "Miinchener  med.  Wochenschrift,"  Sept.  27,  1898. 

||  "Deutsche  med.  Wochenschrift,"  1893,  No.  41. 
**  "Wiener  klin.  Wochenschrift,"  July  12,  1894. 
ft  "Med.  moderne,"  March  25,  1899. 
tJ  "Gaz.  des  Hopitaux,"  1898,  LXXI,  p.  397. 
§§"Brit.  Med.  Jour.,"  1897,  i,  259. 
Illl  Ibid.,  April  17,  1897. 
***  "Brit.  Med.  Jour.,"  April,  3,  1903. 


Bacteriologic  Diagnosis  649 

Meyer  and  Bergell*  prepared  a  serum  by  injecting  horses 
with  a  new  typhoid  toxin.  After  two  years'  treatment 
they  were  able  to  demonstrate  its  value  in  curing  infection 
in  laboratory  animals,  von  Leydenj  speaks  in  favorable 
terms  of  this  serum. 

The  typhoid  immune  (bacteriolytic)  serum  is  specific,  but 
its  action  requires  the  presence  of  additional  complementary 
substance,  and  by  itself  it  is  useless.  Indeed,  it  may  do 
harm  by  causing  the  formation  of  anti-immune  bodies. 

So  far  no  serum  has  been  produced  that  is  of  any  value 
in  therapeutics. 

Bacteriologic  Diagnosis. — There  are  four  means  by 
which  bacteriologic  methods  may  assist  the  clinician  in  corn- 


Fig.   224. — Typhoid  bacilli,  tmag-         Fig.  225. — Typhoid  bacilli,  show- 
glutinated  (Jordan).  ing  typical  clumping  by  typhoid 

serum  (Jordan). 

pleting  the  diagnosis  of  typhoid  fever.     In  the  order  of  their 
general  usefulness  and  practicability  these  are: 

1.  The  Widal  reaction  of  agglutination. 

2.  The  blood-culture. 

3.  The  isolation  of  the  bacillus  from  the  feces. 

4.  Conjunctival  and  dermal  reactions. 

Widal  Reaction  of  Agglutination. — This  very  valuable 
adjunct  to  our  means  of  making  the  diagnosis  of  atypical  and 
obscure  cases  of  typhoidal  infection  was  discovered  in  1896 
when  Widal  and  Griinbaum,  {  working  independently,  ob- 

*  "Med.  Klinik,"  m,  No.  31,  p.  917,  Aug.  4,  1907. 
f'Berl.  klin.  Wochenschrift,"  1907,  No.  18. 
t  "La  Semaine  Medicale,"  1896,  p.  295. 


650  Typhoid  Fever 

served  that  when  blood-serum  from  typhoid  fever  patients 
is  added  to  cultures  of  the  typhoid  bacillus  a  definite  reactive 
phenomenon  occurs.  The  phenomenon,  now  familiarly 
known  as  the  "Widal  reaction,"  consists  of  complete  loss 
of  the  motion  so  characteristic  of  the  typhoid  bacillus,  and 
the  collection  of  the  micro-organisms  into  clusters  or  groups 
— agglutination.  The  bacteria  frequently  appear  shrunken 
and  partly  dissolved. 

The  technic  of  the  test  is  outlined  in  the  section  upon 
Agglutination  (q.  v.).  For  the  use  of  the  practising  physician, 
commercial  houses  now  furnish  various  outfits  known  as 
"agglutometers,"  in  which  are  found  such  simple  apparatus 
and  directions  as  will  enable  those  inexpert  in  laboratory 
manipulations  to  arrive  at  very  accurate  results. 

The  Blood-culture. — The  technic  of  this  operation  is  sim- 
ple. The  skin  of  the  fold  of  the  elbow  is  thoroughly  cleansed, 
a  fillet  put  about  the  arm,  and  as  the  veins  become  promi- 
nent, a  sterile  hypodermic  needle  is  introduced  into  one  and 
about  10  c.c.  of  blood  drawn  into  the  syringe.  Before  clot- 
ting can  take  place,  this  is  discharged  into  a  small  flask  con- 
taining loo  c.c.  of  bouillon,  mixed,  and  stood  away  to  incu- 
bate. After  twenty-four  hours  the  bacilli  can  usually  be 
found  in  pure  culture. 

In  case  the  culture  is  not  pure,  the  typhoid  bacillus  can 
be  separated  from  contaminating  organisms  by  plating. 

The  Isolation  of  the  Bacillus  from  the  Feces. — This 
method  of  making  the  diagnosis  has  practically  been  aban- 
doned because  of  its  uncertainty,  its  cumber someness,  its 
tediousness,  and  because  the  preceding  methods  suffice  in  all 
cases. 

An  excellent  resume  of  the  many  methods  employed  for 
isolating  the  bacillus  from  the  stools  has  been  published  by 
Peabody  and  Pratt,*  and  is  appropriate  reading  for  those 
interested  in  this  subject. 

The  Conjunctival  Reaction. — An  additional  aid  to  the  di- 
agnosis of  typhoid  in  doubtful  cases  based  upon  the  Wolff- 
Eisner- Calmette  reaction  in  tuberculosis  is  the  "ocular  ty- 
phoid reaction"  of  Chantemesse.  f  This  test  consists  in  the 
instillation  into  the  eye  of  a  solution  made  by  extracting  the 
typhoid  bacillus  as  follows:  "Gelatin  plates  covered  with  an 

*  "Boston  Medical  and  Surgical  Journal,"  1907. 

t  "  Deutsche  med.  Wochenschrift,"  1907,  No.  31,  p.  1264. 


Differentiation  of  Typhoid  and  Colon  Bacilli  651 

eighteen-  to  twenty-hour-old  culture  of  virulent  typhoid  bacilli 
were  washed  with  4  to  5  c.c.  of  sterile  water.  The  suspension 
thus  obtained  was  heated  to  60°  C.,  centrifugated,  and  the 
supernatant  fluid  withdrawn.  The  centrifugated  organisms 
were  then  dried  and  triturated.  A  second  suspension  of  these 
broken  up  bacillary  bodies  was  then  made,  and  allowed  to 
stand  for  from  two  to  three  days  at  60°  C.  The  extract 
thus  obtained,  after  removing  the  disintegrated  and  digested 
remnants,  was  precipitated  with  alcohol,  forming  a  fine  coag- 
ulum.  This  was  subsequently  dried  and  powdered  and  dis- 
solved in  sterile  water  in  the  proportion  of  0.02  mg.  to  a 
drop."* 

When  one  drop  of  this  is  placed  upon  the  conjunctiva 
of  a  patient  in  the  early  days  of  typhoid  fever,  diffuse 
redness  increases  and  becomes  marked  in  two  or  three  hours. 
There  is  also  some  feeling  of  heat  in  the  eye.  Tears  flow 
freely,  and  there  is  a  slight  mucopurulent  exudate  in  some 
cases.  The  reaction  persists  about  ten  hours  and  then 
declines,  usually  disappearing  in  twenty-four  hours.  Ham- 
burger f  confirmed  the  results  of  Chantemesse.  It  is  too 
early  to  say  how  useful  the  reaction  is,  but  it  seems  to 
promise  aid  in  diagnosing  difficult  cases. 

Differential  Diagnosis  of  the  Typhoid  and  Colon 
Bacilli. — This  constitutes  the  chief  perplexity  of  bacterio- 
logic  work  with  the  typhoid  bacillus,  and  is  the  great  bugbear 
of  beginners.  A  great  deal  of  energy  has  been  expended  upon 
it,  a  considerable  literature  has  been  written  about  it,  and 
much  still  remains  to  be  learned  by  which  it  may  be  simplified. 

Two  chief  methods  are  in  vogue  at  present: 

1.  The  serum  differentiation. 

2.  The  culture  differentiation. 

Serum  Differentiation. — The  specific  agglutinating  action 
of  experimentally  prepared  serums  can  be  used  to  differentiate 
cultures  of  the  colon,  paracolon,  typhoid,  and  paratyphoid 
bacilli,  the  typhoid  bacilli  alone  exhibiting  the  specific  effect 
of  the  typhoid  serum.  This  is  a  very  reliable  means  of  differ- 
entiation when  the  cultures  have  already  been  isolated.  The 
method  is  described  under  the  heading  "Agglutination,"  in 
the  section  devoted  to  the  "Special  Phenomenon  of  Infection 
and  Immunity." 

*  See  Hamburger,  "Jour.  Amer.  Med.  Assoc.,"  L,  17,  p.  1344,  April  25, 
1908. 

f  Loc.  cit. 


652  Typhoid  Fever 

Richardson*  has  found  it  very  convenient  to  saturate  filter-paper 
with  typhoid  serum,  dry  it,  cut  into  0.5  cm.  squares,  and  keep  it  on 
hand  in  the  laboratory  for  the  purpose  of  making  this  differentiation. 
To  make  a  test,  one  of  these  little  squares  is  dropped  in  0.5  c.c.  of  a 
twenty-four-hour-old  bouillon  culture  of  the  suspected  bacillus  and 
allowed  to  stand  for  five  minutes.  A  drop  .of  the  fluid  placed  upon 
a  slide  and  covered  will  then  show  typical  agglutinations  if  the  culture 
be  one  of  the  typhoid  fever  bacillus.  In  a  second  mention  of  this 
methodf  he  has  found  its  use  satisfactory  in  practice  and  the  paper 
serviceable  after  fourteen  months'  keeping. 

The  Cultural  Differentiation.— When  the  typhoid  bacilli 
are  to  be  isolated  from  the  blood  of  living  patients,  they  are 
so  likely  to  be  obtained  in  pure  culture  that  little  trouble  is 
experienced.  If  they  are  to  be  isolated  from  the  pus  of  a 
posttyphoidal  abscess,  or  from  viscera  at  autopsy,  from 
water  suspected  of  pollution,  and  especially  when  they  are  to 
be  isolated  from  the  intestinal  contents,  with  its  rich  bacterial 
flora,  the  matter  becomes  progressively  complicated. 

As  the  colonies  of  the  typhoid  bacilli  closely  resemble  those 
of  Bacillus  coli,  etc.,  special  media  have,  from  time  to  time, 
been  devised  for  the  purpose  of  emphasizing  such  differences 
as  rapidity  of  growth,  acid  production,  etc.  Thus,  Eisner  t 
has  suggested  the  employment  of  a  special  medium  made  as 
follows : 

One  kilogram  of  grated  potatoes  (the  small  red  German  potatoes 
are  best)  is  permitted  to  macerate  over  night  in  i  liter  of  water.  The 
juice  is  carefully  pressed  out  and  filtered  cold,  to  get  rid  of  as  much  starch 
as  possible.  The  filtrate  is  boiled  and  again  filtered.  The  next  step  is  a 
neutralization,  for  which  Eisner  used  litmus  as  an  indicator,  and  added 
2.5  to  3  c.c.  of  a  TV  normal  sodium  hydrate  solution  to  each.,10  c.c.  of  the 
juice.  Abbott  prefers  to  use  phenolphthalein  as  an  indicator.  The 
final  reaction  should  be  slightly  acid.  Ten  per  cent,  of  gelatin  (no  pep- 
tone or  sodium  chlorid)  is  dissolved  in  the  solution,  which  is  boiled,  and 
must  then  be  again  neutralized  to  the  same  point  as  before.  After  filtra- 
tion the  medium  receives  the  addition  of  i  per  cent,  of  potassium  iodid; 
then  it  is  filled  into  tubes  and  sterilized  like  the  ordinary  culture-media. 

When  water  or  feces  suspected  of  containing  the  typhoid  bacillus  are 
mixed  in  this  medium  and  poured  upon  plates,  no  bacteria  develop  well 
except  the  typhoid  and  colon  bacilli. 

These,  however,  differ  markedly  in  appearance,  for  the  colon  colonies 
appear  of  the  usual  size  in  twenty-four  hours,  at  which  time  the  typhoid 
bacillus,  if  present,  will  have  produced  no  colonies  discoverable  by  the 
microscope. 

It  is  only  after  forty-eight  hours — long  after  the  colon  colonies  have 
become  conspicuous — that  little  colonies  of  the  typhoid  bacillus  appear 
as  finely  granular,  small,  round,  shining,  dew-like  points,  in  marked  con- 
trast to  their  large,  coarsely  granular  predecessors. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1897,  p.  445. 

f  "Journal  of  Experimental  Medicine,"  May,  1898,  p.  353,  note. 

J  "Zeitschrift  fur  Hygiene,"  xxn,  Heft  i,  1895;  Dec.  6,  1896. 


Differentiation  of  Typhoid  and  Colon  Bacilli  653 

Unfortunately,  many  of  the  small  colonies  that  develop 
in  Eisner's  medium  subsequently  prove  to  be  those  of  the 
colon  bacillus,  and  the  method  is  thus  rendered  unreli- 
able. 

Remy*  prefers  to  make  an  artificial  medium  approxi- 
mating a  potato  in  composition,  but  without  dextrin  or  glu- 
cose. The  composition  is  as  follows : 

Distilled  water 1000.0  grains 

Asparagin 6.0 

Oxalic  acid 0.5  gram 

Lactic  acid 0.15 

Citric  acid 0.15 

Disodic  phosphate 5.0  grams 

Magnesium  sulphate 2.5 

Potassium  sulphate 1.25 

Sodium  chlorid 2.0 

All  the  salts  excepting  the  magnesium  sulphate  are  powdered  in  a 
mortar  and  introduced  into  a  flask  with  the  distilled  water.  Thirty 
grams  of  Witte's  or  Grubler's  peptone  are  then  added  and  the  mixture 
heated  in  the  autoclave  under  pressure  for  one-quarter  hour.  As  soon 
as  removed,  the  contents  are  poured  into  another  flask  into  which  120  to 
150  grams  of  gelatin  had  previously  been  placed.  The  flask  is  shaken  to 
dissolve  the  gelatin,  and  the  contents  then  made  slightly  alkaline  with 
soda  solution.  The  mixture  is  again  heated  in  the  autoclave  at  1 10°  C. 
for  one-quarter  hour,  then  acidified  with  a  one-half  normal  solution  of 
sulphuric  acid,  so  that  10  c.c.  have  an  acidity  neutralized  by  0.2  c.c.  of 
one-half  normal  soda  solution.  This  acidity  is  equal  to  0.5  c.c.  sulphuric 
acid  per  liter.  After  shaking,  place  the  flask  in  a  steam  sterilizer  for  ten 
minutes,  then  filter.  When  filtered,  verify  the  acidity  of  the  medium, 
correcting  if  necessary.  Finally,  add  the  magnesium  sulphate,  dissolve, 
dispense  in  tubes,  and  sterilize  by  the  intermittent  method. 

At  the  moment  of  using,  put  into  each  tube  i  c.c.  of  a  35  per  cent, 
solution  of  lactose  and  o.i  c.c.  of  a  2.5  per  cent,  solution  of  carbolic  acid. 

Upon  this  medium  the  colonies  of  the  typhoid  and  colon 
bacilli  show  marked  differences.  The  colon  colonies  are 
yellowish  brown,  the  typhoid  colonies  bluish  white  and 
small.  Fine  bubbles  of  gas  from  the  fermentation  of  the 
lactose  often  occur  about  the  colon  colonies. 

By  this  method  Remy  was  able  to  isolate  the  typhoid 
bacillus  from  the  stools  in  23  cases  which  he  studied.  He 
believes  that  the  constant  presence  of  the  typhoid  bacillus 
in  the  stools  of  typhoid  fever,  and  its  absence  from  them 
under  all  other  conditions,  is  a  far  more  important  and 
valuable  method  of  diagnosis  than  even  the  Widal  re- 
action. 

*  ''Ann.  de  1'Inst.  Pasteur,"  Aug.,  1900. 


654  Typhoid  Fever 

Wiirtz*  and  Kashidaf  make  the  differential  diagnosis  by 
observing  the  acid  production  of  Bacillus  coli  in  a  medium 
consisting  of  bouillon  containing  i  .5  per  cent,  of  agar,  2  per 
cent,  of  milk-sugar,  i  per  cent,  of  urea,  and  30  per  cent,  of 
tincture  of  litmus.  This  is  the  so-called  litmus-lactose-agar- 
agar.  The  culture-medium  should  be  blue.  When  liquefied, 
inoculated  with  the  colon  bacillus,  poured  into  Petri  dishes, 
and  stood  for  from  sixteen  to  eighteen  hours  in  the  incubator, 
the  blue  color  passes  off  and  the  culture-medium  becomes 
red.  If  a  glass  rod  dipped  in  hydrochloric  acid  be  held 
over  the  dish,  vapor  of  ammonium  chlorid  is  given  off.  The 
typhoid  bacillus  produces  no  acid  in  this  medium,  and  there 
is  consequently  no  change  in  its  color.  Upon  plates  with 
colonies  of  both  bacilli,  the  typhoid  colonies  produce  no 
change  of  color,  while  the  colon  colonies  at  once  redden  the 
surrounding  medium. 

Rothberger  J  first  employed  neutral  red  for  the  differentia- 
tion of  the  typhoid  and  colon  bacilli.  When  grown  in  fluid 
media  containing  it,  the  colon  bacillus  produces  a  yellowish 
fluorescence,  while  the  typhoid  bacillus  does  not  destroy  the 
port  wine  color.  Savage  §  and  Irons  ||  have  made  use  of  the 
color  reaction  for  the  routine  detection  of  the  colon  bacillus 
in  water.  The  best  adaptation  of  the  method  is  by  Stokes,** 
who  adds  it  to  the  various  sugar  bouillons  in  the  proportion 
of  o.i  gram  per  liter,  and  uses  the  medium  in  the  fermen- 
tation tube.  The  colon  bacillus  always  ferments  the  sugars 
and  produces  a  typical  color  reaction. 

Hiss  ft  recommends  the  use  of  two  special  media — one 
for  plates,  the  other  for  tube-cultures.  The  first  consists 
of  5  grams  of  agar-agar,  80  grams  of  gelatin,  5  grams  of 
Liebig's  beef -extract,  5  grams  of  sodium  chlorid,  and  10 
grams  of  glucose  to  the  liter.  The  agar  is  dissolved  in  the 
1000  c.c.  of  water,  to  which  have  been  added  the  beef- 
extract  and  sodium  chlorid.  When  the  agar  is  completely 
melted,  the  gelatin  is  added  and  thoroughly  dissolved  by 

*  "Archiv.  de  med.  Experimentale,"  1892,  iv,  p.  85. 

t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Bd.  xxi,  Nos.  20  and  21, 
June  24,  1897. 

t  "Centralbl.  f.  Bakt.,"  1893,  p.  187. 

§  "Journal  of  Hygiene,"  1901,  i,  p.  437. 

||  Ibid.,  1902,  n,  p.  437. 

**  "Jour,  of  Infectious  Diseases,"  1904,  i,  p.  341. 
ft  "Jour,  of  Experimental  Medicine,"  Nov.,  1897,  vol.  n.  No.  6. 


Differentiation  of  Typhoid  and  Colon  Bacilli  655 

a  few  minutes'  boiling.  The  medium  is  then  titrated  to 
determine  its  reaction,  phenolphthalein  being  used  as  the 
indicator,  and  enough  HC1  or  NaOH  added  to  bring  it  to 
the-  desired  reaction — i.  e.,  a  reaction  indicating  1.5  per 
cent,  of  normal  acid.  To  the  clear  medium  add  one  or  two 
eggs,  well  beaten  in  25  c.c.  of  water;  boil  for  forty-five 
minutes,  and  filter  through  a  thin  layer  of  absorbent  cotton. 
Add  the  glucose  after  clearing. 

This  medium  is  used  in  tubes,  in  which  the  culture  is 
planted  by  the  ordinary  puncture.  The  typhoid  bacillus  alone 
has  the  power  of  uniformly  clouding  this  medium  without 
showing  streaks  or  gas-bubbles. 

The  second  medium  is  used  for  plating.  It  contains  10 
grams  of  agar,  25  grams  of  gelatin,  5  grams  of  beef -extract, 
5  grams  of  sodium  chlorid,  and  10  grams  of  glucose.  The 
method  of  preparation  is  the  same  as  for  the  tube-medium, 
care  always  being  taken  to  add  the  gelatin  after  the  agar 
is  thoroughly  melted,  so  as  not  to  alter  this  ingredient  by 
prolonged  exposure  to  high  temperature.  The  preparation 
should  never  contain  less  than  2  per  cent,  of  normal  acid. 
Of  all  the  organisms  upon  which  Hiss  experimented  with 
this  medium,  Bacillus  typhosus  alone  displayed  the  power  of 
producing  thread- forming  colonies. 

The  colonies  of  the  typhoid  bacillus  when  deep  in  Hiss' 
medium  appear  small,  generally  spheric,  with  a  rough, 
irregular  outline,  and,  by  transmitted  light,  of  a  vitreous 
greenish  or  yellowish-green  color.  The  most  characteristic 
feature  consists  of  well-defined  filamentous  outgrowths, 
ranging  from  a  single  thread  to  a  complete  fringe  about  the 
colony.  The  young  colonies  are,  at  times,  composed  solely 
of  threads.  The  fringing  threads  generally  grow  out  nearly 
at  right  angles  to  the  periphery  of  the  colony. 

The  colonies  of  the  colon  bacillus  appear,  on  the  average, 
larger  than  those  of  the  typhoid  bacillus;  they  are  spheric 
or  of  a  whetstone  form,  and  by  transmitted  light  are  darker, 
more  opaque,  and  less  refractive  than  the  typhoid  colonies. 
By  reflected  light  they  are  pale  yellow  to  the  unaided  eye. 

Surface  colonies  are  large,  round,  irregularly  spreading, 
and  are  brown  or  yellowish-brown  in  color.  Hiss  claims 
that  by  the  use  of  these  media  the  typhoid  bacillus  can 
readily  be  detected  in  typhoid  stools. 

Piorkowski*  recommends  a  culture-medium  composed  of 
*  "Berliner  klin.  Wochenschrift,"  Feb.  13,  1899. 


656  Typhoid  Fever 

urine  two  days  old,  to  which  0.5  per  cent,  of  peptone  and  3.3 
per  cent,  of  gelatin  have  been  added.  Colonies  of  the  ty- 
phoid bacillus  appear  radiated  and  filamentous;  those  of  the 
colon  bacillus,  round,  yellowish,  and  sharply  defined  at  the 
edges.  The  cultures  should  be  kept  at  22°  C.,  and  the  colo- 
nies should  appear  in  twenty-four  hours. 

Adami  and  Chapin*  have  suggested  what  seems  to  be  a 
promising  method  for  the  isolation  of  typhoid  bacilli  from 
water,  making  use  of  the  agglutination  of  the  bacilli  by 
immune  serum.  Two  quart  bottles  (Winchester  quarts) 
are  carefully  sterilized  and  filled  with  the  suspected  water 
with  an  addition  of  25  c.c.  of  nutrient  broth  and  incubated 
for  eighteen  to  twenty-four  hours  at  37°  C.  By  this  time 
the  typhoid  bacillus  grows  abundantly  in  spite  of  the  small 
amount  of  nourishment  the  water  contains.  At  the  end  of 
the  incubation,  10  c.c.  of  the  fluid  is  filled  into  each  of  a 
number  of  long  narrow  (7  mm.)  test-tubes  made  by  sealing 
a  glass  tube  one-half  meter  long  at  one  end.  About  one  inch 
from  the  bottom  the  tube  is  filed  completely  round  so  as  to 
break  easily  at  that  point.  The  different  tubes  next  receive 
additions  of  typhoid  immune  serum  sufficient  to  make  the 
dilutions  i  :  60,  i  :  TOO,  i  1150,  and  i  1200.  If  typhoid 
bacilli  are  present,  within  a  quarter  of  an  hour  beginning 
agglutination  can  be  seen,  and  by  the  end  of  two  to  five  hours 
flocculent  masses  collect  at  the  bottom  of  the  tube,  forming 
a  flocculent  precipitate.  The  next  procedure  should  be 
with  the  tube  showing  agglutination  with  the  greatest  dilu- 
tion, as  the  more  concentrated  preparations  carry  down  not 
only  the  typhoid  bacilli,  but  also  closely  related  organisms. 
After  the  sedimentation  of  the  agglutinated  bacilli  is  com- 
plete, the  tube  is  broken  at  the  file  mark,  and  the  sediment 
contained  in  the  short  tube  washed  with  two  or  three  changes 
of  distilled  water,  being  allowed  to  settle  each  time.  This 
removes  many  of  the  organisms  not  agglutinated.  A  loop- 
ful  of  the  washed  sediment  is  transferred  to  a  tube  of  nutrient 
broth,  and  finally  from  this  tube  plate  cultures  are  made  upon 
Eisner's  or  Hiss's  media. 

A  culture  medium  for  isolating  the  typhoid  bacillus  from 
feces  is  recommended  by  Drigalski-Conradif  and  by  Petko- 
witsch.J  It  is  made  as  follows: 

*  "Journal  of  Medical  Research,"  May,  1904,  vol.  xi,  No.  2,  p.  469. 

t  "Zeitschrift  f.  Hygiene,"  Bd.  xxix. 

t  "Centralbl.  f.  Bakt.,"  etc.,  May  28,  1904,  Bd.  xxxvi,  No.  2,  p.  304. 


Differentiation  of  Typhoid  and  Colon  Bacilli   657 

Horse-meat  infusion  (3  pounds  of  horse  meat 

to  2  liters  of  water) 2  liters 

Witte's  peptone 20  grams 

Nutrose 20  grams 

Sodium  chloride 10  grams 

Agar-agar 60  grams 

Litmus  solution  (Kubel  and  Tiemann) 260  c.c. 

Lactose 30  grams 

Crystal- violet  solution  (0.01  per  cent.) 20  c.c. 

Before  adding  the  crystal- violet  solution  render  feebly  alkaline  to 
litmus  (about  0.04  per  cent,  of  pure  soda). 

Colon  colonies  upon  this  medium  appear  in  fourteen  to  sixteen  hours 
to  be  red  and  opaque.  Typhoid  colonies  blue  or  violet,  transparent 
and  drop-like. 

Beckman  *  modifies  the  preparation,  making  it  as  follows : 

"(a)  Add  i  liter  of  water  to  680  grams  of  finely  chopped  lean  beef 
and  place  in  the  cold  for  twenty-four  hours.  Express  the  juice  and 
make  up  to  i  liter.  Coagulate  the  albumin,  either  by  boiling  for  ten 
minutes  or  by  heating  to  120°  C.  in  the  autoclave.  Filter.  Add  10 
grams  of  Witte's  peptone,  10  grams  of  nutrose,  and  5  grams  of  sodium 
chloride.  Heat  in  the  autoclave  at  a  temperature  of  120°  C.  for  thirty 
minutes,  or  boil  vigorously  for  fifteen  minutes.  Render  slightly  alkaline 
to  litmus-paper.  Filter.  Add  30  grams  of  agar.  Heat  in  the  auto- 
clave at  a  temperature  of  1 20°  C.  for  one-half  hour,  or  heat  over  the  gas- 
flame  until  the  agar  is  dissolved.  Render  slightly  alkaline  to  litmus- 
paper  while  hot,  if  necessary.  Filter  through  glass  wool  into  a  sterile 
vessel. 

"  (6)  To  130  c.c.  of  litmus  solution  (Kubel  and  Tiemann' s)  add  15 
grams  of  chemically  pure  lactose.  Boil  for  ten  minutes. 

"  (c)  Mix  (a)  and  (6)  while  hot.  Render  slightly  alkaline  to  litmus, 
if  necessary.  To  the  mixture  add  2  c.c.  of  hot  sterile  solution  of  10  per 
cent,  sodium  hydrate  in  distilled  water  and  10  c.c.  of  a  fresh  solution  of 
Hochst's  crystal  violet  (o.i  gram  of  crystal  violet  to  100  c.c.  of  sterile 
water) . 

"  The  medium  is  now  poured  into  Petri  dishes  and  is  of  a  deep  pur- 
ple color.  So  much  water  of  condensation  forms  on  the  solidified  surface 
that  it  is  an  advantage  to  use  porous  clay  covers  (Hill)  for  the  Petri 
dishes  instead  of  the  ordinary  glass  covers.  The  medium  keeps  well 
but  dries  up  rapidly." 

A  very  ingenious  method  of  isolating  the  typhoid  and 
colon  bacilli  from  drinking  water  has  been  suggested  by 
Starkey,f  who  uses  a  tubular  labyrinth  of  glass  into  which 
a  nutrient  medium,  ordinary  bouillon  containing  0.05  per 
cent,  of  carbolic  acid,  or  Pariette's  bouillon: 

i.  Measure  out  pure  hydrochloric  acid,  4  c.c.,  and  add  it  to  carbolic 
acid  solution  (5  per  cent.),  100  c.c.  Allowr  the  solution  to 
stand  at  least  a  few  days  before  use. 

*  See  F.  F.  Wesbrook,  "Jour.  Infectious  Diseases,"  May,  1905,  Sup- 
plement, No.  i,  p.  319. 

f  "  Amer.  Jour.  Med.  Sci.,"  July,  1906,  cxxxn,  No.  i,  No.  412,  p.  109. 
42 


658 


Typhoid  Fever 


2.  This  solution  is  added  in  quantities  of  o.  i,  0.2,  and  0.3  c.c. 

(delivered  by  means  of  a  sterile  graduated  pipette  to  tubes, 
each  containing  10  c.c.  of  previously  sterilized  nutrient  bouil- 
lon). 

3.  Incubate  at  37°  C.  for  forty-eight  hours  to  eliminate    contami- 

nated tubes. 

as  recommended  by  Somers.*  In  these  labyrinths  the 
motility  of  the  organisms  will  separate  the  colon  and 
typhoid  bacilli  from  non-motile  organisms,  and  from  those 
less  motile  than  themselves.  The  restraining  medium  pre- 
vents the  ready  growth  of  most 
organisms  except  colon  and 
typhoid  bacilli.  The  anaerobic 
conditions  prevent  the  devel- 
opment of  aerobic  organisms 
which  form  the  majority  of 
bacteria  with  which  one  comes 
in  contact  in  ordinary  bac- 
teriological examinations.  The 
typhoid  bacillus,  being  more 
motile  than  the  colon,  travels 
more  quickly  through  the  coils 
of  the  tube  and  first  arrives 
at  its  end,  where  it  can  be 
found  in  pure  or  nearly  pure 
culture  after  about  forty-eight 
hours. 

Somers  has  improved  the 
instrument  by  bending  it  in  a 
circular  form,  so  that  it  can 

stand  alone,  and  by  adapting  its  size  to  the  Novy  jar,  so 
that  satisfactory  anaerobic  conditions  can  easily  be  attained. 
Hesse f  has  recommended  the  following  medium: 

Agar-agar 5  grams  (4.5  grams  absolutely  dry). 

Witte's  peptone 10 

Liebig's  beef -extract 5      " 

Sodium  chlorid 8.5      " 

Distilled  water. .  .  .  1000      " 


Fig.  226. — Starkey's  labyrinth 
as  modified  by  Somers. 


Dissolve  the  agar-agar  in  500  c.c.  of  the  water  over  a  free  flame, 
making  up  the  loss  by  evaporation.  Dissolve  the  other  ingredient,  in  the 
remaining  500  c.c.  of  water,  heat  until  dissolved,  replacing  the  loss  by 

*  "Trans.  Phila,  Path.  Soc.,"  1906. 

t  "Zeitschrift  fur  Hygiene,"  1908,  i,vmf  441. 


Differentiation  of  Typhoid  and  Colon  Bacilli   659 

evaporation.  Pour  the  two  solutions  together  and  heat  for  thirty  minutes 
and  add  distilled  water  to  replace  loss  by  evaporation.  Filter  through 
cotton  until  clear.  Adjust  reaction  to  i  per  cent,  acidity.  Tube — 10 
c.c.  to  a  tube.  Sterilize  in  the  autoclave. 

The  medium  is  used  for  plating.  The  material  containing 
the  micro-organisms  must  be  so  dilute  that  only  a  few  colonies 
will  develop  upon  the  plates.  The  typhoid  colonies  greatly 
outgrow  the  colon  colonies  and  may  attain  to  a  diameter  of 
several  centimeters.  They  show  a  small  opaque  center  and 
an  opalescent  body  and  appear  circular. 

Capaldi*  recommends  the  following  medium  for  plating 
typhoid  and  colon  colonies: 

Witte's  peptone 20  grams 

Gelatin 10 

Agar-agar 20 

Dextrose  or  mannite 10 

Sodium  chlorid 5 

Potassium  chlorid 5 

Distilled  water 1000 

Dissolve  the  agar  in  500  c.c.  of  water,  the  other  ingredients  in  the 
other  500  c.c.  of  water.  Pour  together,  add  10  c.c.  of  NaOH,  filter,  and 
tube. 


Upon  this  medium  the  typhoid  colonies  are  small,  glisten- 
ing, bluish,  and  translucent.  Colon  colonies  are  larger, 
opaque,  and  brownish. 

Endo  f  recommends  the  employment  of  the  following  me- 
dium upon  which  colonies  of  the  typhoid  bacillus  grow  large 
and  remain  colorless,  while  those  of  the  colon  bacillus  remain 
small  and  red: 

1000  c.c.  of  meat  infusion. 
30  grams  of  agar-agar. 
10  grams  of  peptone  (Witte's). 
5  grams  of  sodium  chlorid. 

Neutralize  and  clear  by  filtration,  then  add  10  c.c.  of  a  10  per  cent, 
solution  of  NaOH  to  alkalinize,  10  grams  of  chemically  pure  lactose  and 
5  c.c.  of  a  filtered,  saturated,  alcoholic  solution  of  fuchsin.  Next  add 
25  c.c.  of  a  10  per  cent,  sodium  sulphate  solution,  by  which  the  intense 
red  given  by  the  fuchsin  is  entirely  bleached  by  the  time  the  agar-agar 
is  cold.  After  adding  the  necessary  reagents  and  while  still  warm  and 
perhaps  red,  tube  the  medium.  The  tubes  should  be  kept  in  the  dark. 

*  "Zeitschrift  fur  Hygiene,"  1896,  xxm,  475. 
t  "Centralbl.  f.  Bakt.,"  etc.,  1904,  xxxv. 


66o  Typhoid  Fever 

Loffler*  has  found  malachite  green  a  very  useful  adjunct 
to  our  means  of  differentiating  the  typhoid  from  other  simi- 
lar bacilli. 

For  the  purpose,  2^  to  3  per  cent,  of  a  2  per  cent,  solution  of  malachite 
green  are  added  to  the  culture-medium.  The  preparation  given  the 
preference  consists  of  i  pound  of  meat  macerated  in  i  liter  of  water, 
neutralized  with  potassium,  with  the  addition  of  2  per  cent,  of  peptone,  5 
per  cent,  of  lactose,  i  per  cent,  of  glucose,  0.5  per  cent,  of  sodium  sul- 
phate, 2  per  cent,  of  nitrate  of  potassium,  and  3  per  cent,  of  a  2  per  cent, 
solution  of  malachite  green. 

In  the  medium  the  ordinary  cocci  and  bacilli  do  not  grow, 
Gartner's  bacillus  and  the  paratyphoid  bacillus  b  leave  the 
medium  clear,  but  grow  as  a  deposit  at  the  bottom  of  the 
tube;  the  typhoid  bacillus  destroys  the  green.  If  agar-agar 
be  added,  the  colonies  are  surrounded  by  a  clear  yellow  zone. 
The  colon  and  other  organisms  grow  slowly  if  at  all. 

Not  many  workers  were  satisfied  with  the  results  obtained 
by  malachite  green,  nor  were  the  results  obtained  uniform. 
A  careful  study  of  the  subject  was  made  by  Peabody  and 
Pratt,  f  who  found  great  differences  in  the  quality  and  reac- 
tions of  different  malachite  greens  in  the  market.  That  with 
which  LofBer  worked  was  commercially  known  as  "  120." 
They  obtained  three  samples  of  this  dye,  which  varied  in 
acidity  between  wide  margins  (0.2-1.0).  Experimenting 
with  the  different  preparations,  they  found  that  the  least 
acid  was  the  most  useful  preparation.  The  success  of  the 
method,  therefore,  depends  upon  the  adjustment  of  the 
concentration  of  the  dye  to  the  reaction  of  the  medium. 
When  this  is  done,  malachite  green  becomes  a  valuable 
adjunct  to  specific  differentiation.  Their  studies  of  the 
media  led  Peabody  and  Pratt  to  the  invention  of  a  new 
method  of  isolating  typhoid  bacilli  from  the  feces.  Instead 
of  employing  malachite  green  agar-agar  directly  for  this 
purpose,  they  first  employ  malachite  green  bouillon  as  an 
"  enriching"  culture,  and  after  eighteen  to  twenty-four  hours' 
growth  in  the  incubator  inoculate  one  or  two  large  (20  cm. 
diameter)  Drigalski-Conradi  plates,  from  which  the  colonies 
can  subsequently  be  picked  out. 

Bile  salts  were  first  employed  in  culture-media  by  Lim- 
bourgt  and  have  been  more  or  less  popular  ever  since, 

|  "Inst.  hyg.  Univers.  Griefswald,"  see  "Bull.  Inst.  Past.,"  iv,  No.  9, 
May  15,  1906,  p.  393. 

*  "Boston  Med.  and  Surg.  Journal,"  Feb.  13,  1908,  CLVIII,  p.  213. 
f  "Zeitschrift  f.  physiol.  Chemie,"  1889,  m,  p.  196. 


Differentiation  of  'Typhoid  and  Colon  Bacilli   66 1 

though  for  differentiation  of  typhoid  and  colon  bacilli  they 
cause  occasional  disappointment. 

Buxton  and  Coleman*  prepare  a  medium  composed  of: 

Ox-bile 900  c.c. 

Glycerin 100  c.c. 

Peptone 20  grams 

This  was  placed  in  a  number  of  100  c.c.  flasks,  sterilized  in 
the  Arnold  sterilizer,  and  employed  chiefly  for  blood-culture. 
The  typhoid  bacillus  grows  well  in  it. 

Jackson  f  prepares  a  medium  for  water  examination  when 
typhoid  and  colon  bacilli  are  suspected.  It  consists  of  un- 
diluted ox-bile  to  which  i  per  cent,  of  peptone  and  i  per  cent, 
of  lactose  are  added.  It  is  filled  into  fermentation-tubes  of 
40  c.c.  capacity  and  sterilized  in  the  Arnold  apparatus.  If 
fresh  ox-bile  cannot  be  secured,  an  1 1  per  cent,  solution  of 
fresh  dry  ox-bile  can  be  made;  10  c.c.  of  suspected  water  or 
milk  are  planted  in  the  tubes  of  this  medium.  The  contained 
micro-organisms  grow  rapidly,  typhoid  bacilli  outgrowing 
all  others,  and  not  fermenting  the  sugar ;  rapid  fermentation 
and  copious  gas-formation  take  place  if  colon  bacilli  are 
present. 

An  excellent  medium  suggested  by  MacConkeyJ  has  the 
following  composition: 

Agar 1.5  grams 

Sodium  taurocholate 0.5  gram 

Peptone 2.0  grams 

Water ico.o  c.c. 

It  is  boiled,  clarified,  and  filtered  as  usual,  then  receives  an  addition 
of  i.o  gram  of  lactose,  is  tubed,  and  then  sterilized  three  times  on  suc- 
cessive days. 

For  determining  fermentation  by  colon  bacilli  the  same 
investigator  advises  a  broth  composed  of: 

Sodium  taurocholate  (pure) 0.5  gram 

Peptone 2.0  grams 

Glucose 0.5  gram 

Water .  100.0  c.c. 

Boil,  filter,  add  sufficient  neutral  litmus,  fill  into  fermentation-tubes, 
and  sterilize  at  100°  C.  Colon  colonies  appear  red;  typhoid,  blue. 

*  "Journal  of  Infectious  Diseases,"  1909,  vi,  No.  2,  p.  194. 
t  "Biological  Studies  of  the  Pupils  of  W.  T.  Sedgwick,"  1906,  Uni- 
versity of  Chicago  Press. 

t  "The  Thompson- Yates  Laboratory  Reports,"  in,  p.  151. 


662  Typhoid  Fever 

In  a  careful  study  of  the  bile-salt  media  MacConkey* 
points  out  an  error,  first  discovered  by  Theobald  Smith,  that 
depends  upon  the  alkali  production  of  the  colon  bacillus  in 
the  absence  of  sugar.  If  too  little  sugar  be  added  to  the 
medium,  the  alkali  production  masks  the  acid  production 
unless  the  oxygen  be  removed,  and  red  colonies  of  the  colon 
bacillus  grown  upon  the  medium  may  in  time  turn  dis- 
tinctly blue.  It  becomes  obvious,  therefore,  that  the 
medium  should  be  as  neutral  as  possible  to  the  indicator 
used.  After  trial  he  found  neutral  red  preferable  to  litmus, 
and  makes  the  medium  as  follows: 

i.  A  stock  solution  is  made: 

Sodium  taurocholate  (commercial  from  ox-bile 

and  neutral  to  neutral  red) 0.5  per  cent. 

Peptone  (White's) 2.0 

Water  (distilled  or  tap) 100.0  c.c. 

(As  calcium  0.03  per  cent,  is  favorable  to  the  growth  of  the 

organisms,  it  should  be  added  if  distilled  water  is  used.) 

The  ingredients  should  be  mixed,  steamed  in  a  steam  sterilizer  for 
one  to  two  hours,  filtered  while  hot,  allowed  to  stand  twenty-four  to 
forty-eight  hours,  then  filtered  cold  through  paper.  A  clear  solution 
should  then  result,  which  will  keep  indefinitely  under  proper  conditions. 
The  various  bile-salt  media  are  prepared  from  this  stock  solution  by 
adding  glucose,  0.5  per  cent. ;  lactose,  i  per  cent. ;  cane-sugar,  i  per  cent. ; 
dulcit,  0.5  per  cent.;  adonit,  0.5  per  cent.,  or  inulin,  i  per  cent.;  and 
neutral  red  (i  per  cent,  solution),  0.25  per  cent.,  distributing  into  fer- 
mentation-tubes and  sterilizing  in  the  steamer  for  fifteen  minutes  on 
each  of  three  successive  days. 

Bile-salt  agar-agar  is  made  by  dissolving  2  per  cent,  of  agar-agar  in 
the  stock  fluid,  either  in  the  steamer  or  in  the  autoclave.  The  mixture 
is  cleared  with  an  egg,  filtered,  neutral  red  added  in  the  same  proportion 
as  for  the  broth,  and  distributed  into  flasks  in  quantities  of  80  c.c. 
When  required  for  use,  the  fermentable  substance  is  added  to  the  agar 
in  the  flask,  and  the  whole  placed  in  a  water-bath  or  steamer  (care  must 
be  taken  not  to  heat  either  the  fluid  or  solid  medium  beyond  100°  C.). 
When  melted,  the  agar  preparation  is  poured  into  Petri  dishes,  allowed 
to  solidify,  and  then  dried  in  an  incubator  or  warm  room,  the  plate  being 
placed  upside  down  with  the  bottom  detached  and  propped  up  on  the 
edge  of  the  cover.  It  is  necessary  that  the  surface  of  the  agar-agar 
should  not  be  too  wet,  lest  the  colonies  become  confluent,  nor  too  dry, 
lest  the  growth  be  stunted.  Inoculations  are  made  by  placing  a  loop- 
ful  of  the  material  to  be  examined  on  the  center  of  one  plate,  and 
rubbed  over  the  surface  with  a  bent  glass-rod;  the  same  rod,  without 
recharging,  being  used  to  inoculate  the  surface  of  two  other  plates. 
The  plates  are  then  incubated  upside  down.  The  colonies  of  the  colon 
bacillus  appear  yellow. 

*  ''Journal  of  Hygiene,"  1908,  vm,  p.  322. 


Bacilli  Resembling  the  Typhoid  Bacillus       663 

BACILLI  RESEMBLING  THE  TYPHOID  BACILLUS. 

Bacillus  typhosus  is  one  of  a  group  of  organisms  pos- 
sessing a  considerable  number  of  common  characteristics, 
each  member  of  which,  however,  can  be  differentiated  by 
some  one  fairly  well-marked  peculiarity.  At  one  end  of 
the  series  is  the  typhoid  bacillus,  which  we  conceive  to  be 
devoid  of  the  power  to  liquefy  gelatin,  ferment  sugars,  form 
indol,  coagulate  milk,  or  progressively  form  acids.  At  the 
other  extreme  stands  Bacillus  coli,  an  organism  whose 
typical  representatives  coagulate  milk,  form  indol,  ferment 
dextrose,  lactose,  saccharose,  and  maltose  with  the  forma- 
tion of  hydrogen  'and  carbon  dioxid  in  the  proportion  of 

H       2^ 

coT  ~  i- 

Between  these  extremes  are  numerous  organisms  known 
as  "intermediates."  It  is  usually  a  simple  matter  to  dif- 
ferentiate these  forms  from  the  typical  species  at  the  two 
ends  of  the  series,  but  it  is  quite  difficult  to  differentiate 
them  from  one  another.  Whether  they  are  of  sufficient 
importance  to  make  it  worth  while  to  pay  much  attention 
to  them  is,  as  yet,  uncertain;  and,  indeed,  we  do  not  know 
whether  they  are  to  be  regarded  as  variations  from  the 
type  species  or  separate  and  distinct  organisms.  The  fact 
that  some  of  them  are  associated  with  serious  and  fatal 
disorders — paracolon  bacillus  and  bacillus  of  psittacosis — 
proves  them,  at  least,  to  be  important. 

In  his  careful  review  of  the  intermediate  forms  thus  far 
described,  Buxton*  summarizes  the  main  points  of  difference 
as  follows: 

B.  coli  com- 

munis.       Intermediates.     B.  typhosus. 

Coagulation  of  milk -+- 

Production  of  indol -j- 

Fermentation  of  lactose  with  gas ....  -j- 

Fermentation  of  glucose  with  gas.  .  .  -j-  + 

Agglutination  by  typhoid  serum ....  -f- 

The  characteristics  of  the  three  groups  as  shown  by  the 
fermentation-test  stand  thus:f 

Gas  upon        Gas  upon  Gas  upon 

dextrose.          lactose.  saccharose. 

Bacillus  typhosis 

Intermediates + 

Bacillus  coli  communis -j-  -f- 

Bacillus  coli  communior -j-  -j-  + 

c  "Journal  of  Medical  Research,"  vol.  vm,  No.  i,  June,  1902,  p.  201. 
t  Hiss  and  Zinsser,  "Text-book  of  Bacteriology,"  1910,  p.  429. 


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Bacilli  Resembling  the  Typhoid  Bacillus       665 

Buxton  finds  those  pathogenic  for  man  clinically  divisible 
into  three  groups,  as  follows: 

(a)  The  Meat- poisoning  Group. — This  includes   Bacillus 
enteritidis  of  Gartner  and  others.     The  symptoms  begin  soon 
after  eating  the  poisonous  meat,   and  are  toxic.     Bacilli 
quickly  invade  the  body.     The  illness  continues  four  or 
five  days,  after  which  recovery  is  quick.     In  a  few  cases 
death  has  occurred  on  the  second  or  third  day. 

(b)  The  Pneumonic  or  Psittacosis  Group. — Psittacosis  is 
an  epidemic  infectious  disease  with  pneumonic  symptoms 
and  a  high  mortality.     Its  origin  has  been  traced  to  dis- 
eased  parrots,   and   from   them   Nocard   isolated   Bacillus 
psittacosis,  supposed  to  be  the  cause  of  the  disease  in  man. 
Later  epidemics  were  studied  by  Achard  and  Bensaude. 

(c)  The  Typhoidal  Group. — The  organisms  to  be  included 
in  this  group  occasion  symptoms  closely  resembling  typhoid 
fever,    though   they   differ   biologically   from   the   typhoid 
bacillus,  and  do  not  agglutinate  with  typhoid  serums. 

It  is  thus  evident  that  some  of  the  intermediates  occasion 
symptoms  resembling  typhoid  fever,  while  others  occasion 
symptoms  widely  differing  from  it.  It  is  suggested  that  to 
the  former  the  term  paratyphoid  bacilli  be  applied,  while  the 
latter  are  known  as  paracolon  bacilli. 

Although  Achard  and  Bensaude,*  and  Johnson,  Hewlett, 
and  Longcope  f  have  studied  the  paratyphoid  infections, 
Gwyn,t  Libman,§  and  others  the  paracolon  bacilli,  and 
Gushing  ||  and  Durham  **  have  made  comparative  studies 
of  the  members  of  the  group,  it  is  still  too  soon  to  regard 
the  knowledge  attained  sufficient  to  warrant  particular 
mention  of  the  various  intermediate  and  related  organisms 
in  a  work  of  this  kind.  In  the  following  pages,  therefore, 
attention  will  be  devoted  only  to  the  more  important 
organisms  of  the  group  and  to  a  few  belonging  to  offshoots 
from  the  parent  stem. 

*  "Soc.  Med.,"  Nov.,  1896. 

t  "  Amer.  Jour.  Med.  Sci.,"  Aug.,  1902. 

J  "Johns  Hopkins  Bulletin,"  vol.  ix,   1898. 

§  "Journal  of  Medical  Research,"  1902,  p.  168. 

||  "Johns  Hopkins  Bulletin,"  vol.  xi,  1900. 

**  "Journal  of  Experimental  Medicine,"  vol.  v,  p.  353,  1901. 


666  Bacillus  Coli 

BACILLUS  COLI  COMMUNIS  (ESCHERICH). 

General  Characteristics. — A  motile,  flagellated,  non-sporogenous, 
aerobic  and  optionally  anaerobic,  non-chromogenic,  non-liquefying, 
aerogenic,  saprophytic,  occasionally  pathogenic  bacillus,  staining  by 
the  ordinary  methods,  but  not  by  Gram's  method.  It  produces  indol, 
coagulates  milk,  and  produces  acids  and  gases  from  dextrose,  lactose, 
and  sucrose. 

This  micro-organism  was  first  isolated  from  human  feces  by 
Emmerich,*  in  1895,  who  thought  it  to  be  the  specific  cause 
of  Asiatic  cholera,  and  called  it  Bacillus  neapolitanus. 
Many  investigators  have  since  studied  its  peculiarities,  until 
it  has  become  one  of  the  best  known  bacteria. 


Fig.  227. — Bacillus  coli  (Migula). 

Distribution. — It  is  habitually  present  in  the  feces  of 
animals,  and  in  water  and  soil  contaminated  with  them. 
Soon  after  birth  the  organism  finds  its  way  into  the  alimen- 
tary canal  and  permanently  establishes  itself  in  the  intes- 
tine, where  it  can  be  found  in  great  numbers  throughout 
the  entire  life  of  the  individual.  It  is  almost  certainly 
identical  with  Bacillus  pyogenes  foetidus  of  Passet,  and  so 
closely  resembles  B.  acidi  lactici  that  Prescott  f  believes 
them  to  be  identical.  It  may  also  be  identical  with  Ba- 
cillus lactis  aerogenes,  Bacillus  cavicida,  and  other  described 
species. 

*"  Deutsche  med.  Wochenschrift,"   1885,  No.  2. 

t  Society  of  American  Bacteriologists,  Dec.  31,  1902. 


Bacilli  Resembling  the  Typhoid  Bacillus       667 

Morphology. — The  bacillus  is  rather  variable,  both  size 
and  form  depending  to  a  certain  extent  upon  the  culture 
medium  on  which  it  grows.  It  measured  about  1-3  X  0.4- 
0.7  fj..  It  usually  occurs  in  the  form  of  short  rods,  but 
coccus-like  individuals  and  elongate  individuals  may  be 
found  in  the  same  culture.  The  bacilli  are  usually  separate 
from  one  another,  though  occasionally  joined  in  pairs,  are 
actively  motile,  and  provided  with  flagella,  which  are  vari- 
able in  number,  usually  from  four  to  a  dozen.  The  organ- 
isms from  some  cultures  swim  actively,  even  when  the  cul- 
ture is  some  days  old ;  others  are  sluggish  even  when  young 


Fig.  228. — Bacillus  coli  communis;  superficial  colony  two  days  old 
upon  a  gelatin  plate.     X  21  (Heim). 

and  actively  growing,  and  still  other  cultures  consist  of 
bacilli  that  scarcely  move  at  all.  It  forms  no  endospores. 

Staining. — The  bacillus  stains  well  with  the  aqueous 
solutions  of  the  anilin  dyes,  but  not  by  Gram's  method. 

Cultivation. — It  is  readily  cultivated  upon  the  ordinary 
media. 

Colonies. — Upon  gelatin  plates  the  colonies  are  visible 
in  twenty-four  hours.  Those  situated  below  the  surface 
appear  round,  yellow-brown,  and  homogeneous.  As  they 
increase  in  size  they  become  opaque.  The  superficial 
colonies  are  larger  and  spread  out  upon  the  surface.  The 
edges  are  dentate  and  slightly  resemble  grape-vine  leaves, 
often  showing  radiating  ridges  suggestive  of  the  veins  of  a 
leaf.  They  may  have  a  slightly  concentric  appearance. 
The  colonies  rapidly  increase  in  size  and  become  more  and 
more  opaque.  The  gelatin  is  not  liquefied. 


668  Bacillus  Coli 

Gelatin  Punctures. — Development  in  gelatin  punctures 
occurs  upon  the  surface,  and  also  in  the  needle's  track,  caus- 
ing the  formation  of  a  nail-like  growth.  The  head  of  the 
nail  may  reach  the  walls  of  the  test-tube.  No  gas  is  formed 
in  ordinary  gelatin,  but  should  any  dextrose  be  present  in  it, 
gas-production  may  occur  and  irregularly  break  up  the  me- 
dium. The  gelatin  may  become  slightly  clouded  as  the 
bacilli  grow,  but  is  not  liquefied. 

Agar-agar. — Upon  agar-agar  along  the  line  of  inocula- 
tion a  grayish- white,  translucent,  smeary  growth  devoid  of 
any  characteristics  takes  place.  The  entire  surface  of  the 
culture-medium  is  never  covered,  the  growth  remaining  con- 
fined to  the  inoculation  line,  except  where  the  moisture  of 
condensation  allows  it  to  spread  out  at  the  bottom.  Kruse 
says  that  crystals  may  form  in  old  cultures. 

Bouillon. — Bouillon  is  densely  clouded  by  the  growth  of 
the  bacteria,  a  delicate  pellicle  at  times  forming  upon  the  sur- 
face. There  is  usually  considerable  sediment  in  the  culture. 

Potato. — Upon  potato  the  growth  is  luxuriant.  The 
bacillus  forms  a  yellowish -brown,  glistening  layer  spreading 
from  the  line  of  inoculation  over  about  one-half  to  two- 
thirds  of  the  potato.  The  color  varies  considerably,  some- 
times being  pale,  sometimes  quite  brown,  sometimes  green- 
ish. It  cannot,  therefore,  be  taken  as  a  characteristic  of 
much  importance.  The  growth  on  potato  may  be  almost 
invisible. 

Milk. — In  milk  rapid  coagulation  and  acidulation  occur, 
with  the  evolution  of  gas.  The  culture  gives  off  a  fecal  odor. 
Litmus  added  to  the  culture  media  is  first  reddened,  then 
decolorized  by  the  bacilli. 

Vital  Resistance. — It  is  quite  resistant  to  antiseptics 
and  germicides,  and  grows  in  culture  media  containing 
from  0.1-0.2  per  cent,  of  carbolic  acid.  It  is,  however, 
easily  killed  by  heat,  and  is  destroyed  by  exposure  to  60° 
C.  for  ten  minutes. 

Metabolic  Products. — Wiirtz  found  that  Bacillus  coli 
produced  ammonia  in  culture  media  free  from  sugar,  and 
thus  caused  an  intense  alkaline  reaction  in  the  culture 
media.  The  cultures  usually  give  off  an  odor  that  varies 
somewhat,  but  is,  as  a  rule,  unpleasant. 

Nitrates  are  reduced  to  nitrites  by  the  growth  of  the 
bacillus. 

In  bouillon  containing  i  per  cent,  of  dextrose,  lactose, 
levulose,  galactose,  and  mannite,  the  colon  bacillus  splits  up 


Bacilli  Resembling  the  Typhoid  Bacillus       669 

the  sugar,  liberating  CO2  and  H,  the  gas  formula  being 
^  =  ~.  This  gas  formula  is  very  constant  for  the  micro- 
organisms of  the  colon  group  and  forms  one  of  their  most 
important  differential  characteristics.  In  sugar-containing 
bouillon,  acetic,  lactic,  and  formic  acids  are  produced.  It 
does  not  ferment  saccharose.  When  a  similar  bacillus  is 
found  to  ferment  saccharose  it  is  best  regarded  as  a  sub- 
species or  separate  type,  for  which  Dunham  has  introduced 
the  name  Bacillus  coli  communior. 

The  bacillus  requires  very  little  nutriment.  It  grows  in 
Uschinsky's  asparagin  solution,  and  is  frequently  found  living 
in  river  and  well  waters. 

Indol  is  formed  in  both  bouillon  and  peptone  solutions, 
but  phenol  is  not  produced.  The  presence  of  indol  is  best 
determined  by  Salkowski's  method  (q.  V.). 

Toxic  Products. — Vaughan  and  Cooley*  have  shown 
that  the  toxin  of  the  colon  bacillus  is  contained  in  the 
germ-cell  and  under  ordinary  conditions  does  not  diffuse 
from  it  into  the  culture  medium.  The  toxin  may  be  heated 
in  water  to  a  very  high  temperature  without  injuring  its 
poisonous  nature.  They  have  devised  an  apparatus  in 
which  enormous  cultures  can  be  prepared  and  the  bacteria 
pulverized. f  Of  such  a  preparation  0.0002  gram  will  kill  a 
2oo-gram  guinea-pig. 

Pathogenesis. — The  bacillus  begins  to  penetrate  the 
intestinal  tissues  almost  immediately  after  death,  and  is  the 
most  frequent  contaminating  micro-organism  met  with  in 
cultures  made  at  autopsy.  It  may  spread  by  direct  con- 
tinuity of  tissue,  or  via  the  blood-vessels. 

Although  under  normal  conditions  a  saprophyte,  the 
colon  bacillus  is  not  infrequently  found  in  the  pus  in  sup- 
purations connected  with  the  intestines — as,  for  example, 
appendicitis — and  sometimes  in  suppurations  remote  from 
them. 

In  intestinal  diseases,  such  as  typhoid,  cholera,  and 
dysentery,  the  bacillus  not  only  seems  to  acquire  an  unusual 
degree  of  virulence,  but  because  of  the  existing  denudation 
of  mucous  surfaces,  etc.,  finds  it  easy  to  enter  the  general 
system,  with  the  formation  of  remote  secondary  suppura- 
tive  lesions  in  which  it  is  the  essential  factor.  When  ab- 
sorbed from  the  intestine,  it  frequently  enters  the  kidney 

*  "Jour.  Amer.  Med.  Assoc.,"  1901;  "American  Medicine,"  1901. 
f  "Trans.  Assoc.  Amer.  Phys.,"  1901. 


670  Bacillus  Coli 

and  is  excreted  with  the  urine,  causing,  incidentally,  local 
inflammatory  areas  in  the  kidney,  and  occasionally  cystitis. 
A  case  of  urethritis  is  reported  to  have  been  caused  by  it. 

In  infants  cholera  infantum  may  not  infrequently  be 
caused  by  the  colon  bacillus,  though  probably  in  this  disease 
other  bacteria  play  an  important  role  (B.  dysenteriae?). 

The  bile-ducts  are  sometimes  invaded  by  the  bacillus, 
which  may  lead  to  inflammation,  obstruction,  suppuration, 
or  calculus  formation. 

The  colon  bacillus  has  also  been  met  with  in  puerperal 
fever,  Winckel's  disease  of  the  newborn,*  endocarditis,  men- 
ingitis, liver-abscess,  bronchopneumonia,  pleuritis,  chronic 
tonsillitis,  urethritis,  and  arthritis. 

An  interesting  summary  of  the  pathogenic  effect  of  Bacillus 
coli  can  be  found  in  Rolleston's  paper  in  the  "British  Medi- 
cal Journal"  for  Nov.  4,  1911,  p.  1186. 

In  a  certain  number  of  cases  general  hemic  infection  may 
be  caused  by  Bacillus  coli.  In  1909  Jacob t  published  an 
analysis  of  39  such  cases,  and  in  1910  Draper!  increased  the 
number  to  43.  Wiens§  also  reported  6  cases  and  Maher||  i 
case,  so  that  the  total  now  stands  50. 

Virulence. — It  is  a  question  whether  the  colon  bacillus  is 
always  virulent,  or  whether  it  becomes  so  under  abnormal 
conditions.  Klencki**  found  it  very  virulent  in  the  ileum, 
and  less  so  in  the  colon  and  jejunum  of  dogs.  He  also 
found  that  the  virulence  was  greatly  increased  in  a  strangu- 
lated portion  of  intestine.  Dreyfus  ft  found  that  the  colon 
bacillus  as  it  occurs  in  normal  feces  is  not  virulent.  Most 
experimenters  believe  that  pathologic  conditions,  such  as 
disease  of  the  intestine,  strangulation  of  the  intestine,  etc., 
increase  its  virulence. 

Frequent  transplantation  lessens  the  virulence  of  the 
bacillus;  passage  through  animals  increases  it. 

It  has  been  observed  that  cultures  of  the  bacillus  obtained 
from  cases  of  cholera,  cholera  nostras,  and  other  intestinal 
diseases  are  more  pathogenic  than  those  obtained  from 
normal  feces  or  from  pus. 

"Kamen-Ziegler's  Beitrage,"  1896,  14. 

t  "Deutsch.  Archiv.  f.  Klin.  Med.,"  1909,  xcvii,  303. 

t  "Bull,  of  the  Ayer  Clin.  Lab.  of  the  Penna.  Hosp.,"  1910,  No.  6,  p.  21. 

§  "Munch,  med.  Woch.,"  1909,  LVI,  962. 

||  "Med.  Record,"  1909,  LXXV,  482. 
**  "Ann.  de  1'Inst.  Pasteur,"  1895,  No.  9. 
ft  "Centralbl.  f.  Bakt.,"  etc.,  xvi,  p.  581 


Bacilli  Resembling  the  Typhoid  Bacillus      671 

Adelaide  Ward  Peckham,*  in  an  elaborate  study  of  the 
"Influence  of  Environment  on  the  Colon  Bacillus,"  con- 
cludes that  while  the  conditions  of  nutrition  and  develop- 
ment in  the  intestine  seem  to  be  most  favorable,  the  colon 
bacillus  is  ordinarily  not  virulent.  She  says: 

"Its  first  force  is  spent  upon  the  process  of  fermentation,  and  as 
long  as  opportunities  exist  for  the  exercise  of  this  function  the  affinities 
of  this  organism  appear  to  be  strongest  in  this  direction. 

"Moreover,  the  contents  of  the  intestine  remain  acid  until  they 
reach  the  neighborhood  of  the  colon,  and  by  that  time  the  tryptic 
peptones  have  been  formed  and  absorbed  to  a  great  extent. 

"During  the  process  of  inflammation  in  the  digestive  tract  a  very 
different  condition  may  exist.  The  peptic  and  tryptic  enzymes  may 
be  partially  suppressed.  Fermentation  of  carbohydrates  and  proteid 
foods  then  begins  in  the  stomach,  and  continues  after  the  mass  of 
food  is  passed  on  into  the  intestine.  The  colon  bacillus  cannot,  there- 
fore, spend  its  force  upon  fermentation  of  sugars,  because  they  are 
already  broken  up  and  an  alkaline  fermentation  of  the  proteids  is  in 
progress.  It  also  cannot  form  peptones  from  the  original  proteids, 
for  it  does  not  possess  this  property,  and  unless  trypsin  is  present  it 
must  be  dependent  upon  the  proteolytic  activity  of  other  bacteria  for 
a  suitable  form  of  proteid  food.  Perhaps  these  bacteria  form  an  albu- 
minate  molecule  which,  like  leucin  and  tyrosin,  cannot  be  broken  up 
into  indol,  and  thus  there  might  be  caused  an  important  modification 
of  the  metabolism  of  the  colon  bacillus,  which  might  have  either  an 
immediate  or  remote  influence  upon  its  acquisition  of  disease-producing 
properties,  for  our  own  experiments  indicate  that  the  power  to  form 
indol,  and  the  actual  forming  of  it,  are  to  some  extent  an  indication 
of  the  possession  of  pathogenesis." 

For  the  laboratory  animals  the  colon  bacillus  is  patho- 
genic in  varying  degree.  Intraperitoneal  injections  into 
mice  cause  death  in  from  one  to  eight  days  if  the  culture  be 
virulent.  Guinea-pigs  and  rabbits  also  succumb  to  intra- 
peritoneal  and  intravenous  injection.  Subcutaneous  injec- 
tions are  of  less  effect,  and  in  rabbits  seem  to  produce 
abscesses  only. 

When  injected  into  the  abdominal  cavity,  the  bacilli 
set  up  a  sero-fibrinous  or  purulent  peritonitis,  and  are  very 
numerous  in  the  abdominal  fluids. 

Cumston,  f  from  a  careful  study  of  thirteen  cases  of  summer 
infantile  diarrheas,  comes  to  the  following  conclusions: 

Bacterium  coli  seems  to  be  the  pathogenic  agent  of  the  greater 
number  of  summer  infantile  diarrheas. 

The  organism  is  often  associated  with  Streptpcoccus  pyogenes. 

The  virulence,  more  considerable  than  in  the  intestine  of  a  healthy 
child,  is  almost  always  in  direct  relation  to  the  condition  of  the  child 

*  "Journal  of  Experimental  Medicine,"  Sept.,  1897,  vol.  n,  No.  4, 
P-  549- 

t  "International  Medical  Magazine,"  Feb.,  1897. 


672  Bacillus  Coli 

at  the  time  the  culture  is  taken,  and  does  not  appear  to  be  propor- 
tionate to  the  ulterior  gravity  of  the  case. 

The  mobility  of  Bacterium  coli  is,  in  general,  proportionate  to  its 
virulence.  The  jumping  movement,  nevertheless,  does  not  correspond 
to  an  exalted  virulence  in  comparison  with  the  cases  in  which  the 
mobility  was  very  considerable,  without  presenting  these  jumping 
movements. 

The  virulence  of  Bacterium  coli  found  in  the  blood  and  other  organs 
is  identical  with  that  of  Bacterium  coli  taken  from  the  intestines  of  the 
individual. 


Lesage,*  in  studying  the  enteritis  of  infants,  found  that 
in  40  out  of  50  cases  depending  upon  Bacillus  coli  the  blood 
of  the  patient  agglutinated  the  cultures  obtained,  not  only 
from  his  own  stools,  but  from  those  of  all  the  other  cases. 
From  this  uniformity  of  action  Lesage  suggests  that  the 
colon  bacilli  in  these  cases  are  all  of  the  same  species. 

The  agglutinating  reaction  occurs  only  in  the  early  stages 
and  acute  forms  of  the  disease. 

Immunization. — It  is  not  difficult  to  immunize  an 
animal  against  the  colon  bacillus.  Loffler  and  Abel  im- 
munized dogs  by  progressively  increased  subcutaneous 
doses  of  live  bacteria,  grown  in  solid  culture  and  suspended 
in  water.  The  injections  at  first  produced  hard  swellings. 
The  blood  of  the  immunized  animals  possessed  an  active 
bactericidal  effect  upon  the  colon  bacteria.  The  serum 
was  not  in  the  correct  sense  antitoxic. 

Differential  Diagnosis. — This  problem  is  considered  at 
greater  length  under  the  heading  "Cultural  Differentiation 
of  the  Bacillus  Typhosus"  (q.v.).  For  the  recognition  of  the 
colon  bacillus  the  most  important  points  are  the  motility, 
the  indol-formation,  the  milk-coagulation,  and  the  active 
gas-production.  As,  however,  most  of  these  features  are 
shared  by  other  bacteria  to  a  greater  or  less  degree,  the  most 
accurate  differential  point  is  the  immunity  reaction  with  the 
serum  of  an  immunized  animal,  which  protects  susceptible 
animals  from  the  effects  of  inoculation,  and  produces  a  simi- 
lar agglutinative  reaction  to  that  observed  in  connection  with 
the  blood  and  serum  of  typhoid  patients,  convalescents,  and 
immunized  animals. 

The  fact  that,  with  rare  exceptions,  the  typhoid  serum 
produces  a  specific  reaction  with  the  typhoid  bacillus,  and 
the  colon  serum  with  the  colon  bacillus,  should  be  the  most 
important  evidence  that  they  are  entirely  different  species. 

What  is  commonly  known  as  Bacillus  coli  communis  is, 
no  doubt,  not  a  single  species,  but  a  group  of  bacilli  too 
*  "La  Semaine  Medicale,"  Oct.  20,  1897. 


Bacilli  Resembling  the  Typhoid  Bacillus       673 

similar  to  be  differentiated  into  groups,  types,  or  families 
by  our  present  methods. 

In  order  to  establish  a  type  species  of  Bacillus  coli  com- 
munis,  Smith  *  says : 

"I  would  suggest  that  those  forms  be  regarded  as  true  to  this  species 
which  grow  on  gelatin  in  the  form  of  delicate  bluish  or  more  opaque, 
whitish  expansions  with  irregular  margin,  which  are  actively  motile 
when  examined  in  the  hanging  drop  from  young  surface  colonies  taken 
from  gelatin  plates,  which  coagulate  milk  within  a  few  days ;  grow  upon 
potato,  either  as  a  rich  pale  or  brownish-yellow  deposit,  or  merely 
as  a  glistening,  barely  recognizable  layer,  and  which  give  a  distinct 
indol  reaction.  Their  behavior  in  the  fermentation-tube  must  conform 
to  the  following  scheme: 

"  Variety  a: 

"One  per  cent,  dextrose-bouillon  (at  37°  C.).  Total  gas  approx- 
imately i;  H —  COa  =  approximately  2  :  1 ;  reaction  strongly  acid. 

"One  per  cent,  lactose-bouillon:  as  in  dextrose-bouillon  (with  slight 
variations). 

"One  per  cent,  saccharose-bouillon;  gas-production  slower  than 
the  preceding,  lasting  from  seven  to  fourteen  days.  Total  gas  about 
$;  H  —  CO2  =  nearly  3:2.  The  final  reaction  in  the  bulb  may  be 
slightly  acid  or  alkaline,  according  to  the  rate  of  gas-production  (B.  coli 
communior,  Dunham). 

"  Variety  /3: 

"The  same  in  all  respects,  excepting  as  to  its  behavior  in  saccharose- 
bouillon;  neither  gas  nor  acids  are  formed  in  it." 

DIFFERENTIAL  CHARACTERISTICS. 
TYPHOID  BACILLUS.  COLON  BACILLUS. 

Bacilli  usually  slender.  Bacilli  a  little  thicker  and  shorter. 

Flagella  numerous  (10-20),  long,  Flagella  fewer  (8-10)  (peritricha). 

and  wavy  (peritricha). 

Growth  not  very  rapid,  not  par-  Growth  rapid  and  luxuriant.  This 

ticularly  luxuriant.  character  is  by  no  means  con- 
stant. 

Upon    Eisner's,    Hiss',    Piorkow-  Upon    Eisner's,    Hiss',    Piorkow- 

ski's,    and    other   media    gives  ski's,  and    other    media    gives 

characteristic  appearances.  characteristic  appearances. 

Upon  fresh  acid   potato  the  so-  Upon    potato  a  brownish-yellow 

called  "invisible  growth"  form-  distinct  pellicle. 

erly  thought  to  be  differential. 

Acid-production  in  whey  not  ex-  Acid-production       well      marked 

ceeding  3  per  cent.    Sometimes  throughout. 

slight  in  ordinary  media,    and 

succeeded  by  alkali-production. 

Grows  in  media  containing  sugars  Fermentation    with    gas-produc- 

without  producing  any  gas.  tion   well  marked  in  solutions 

containing  dextrose,  lactose, 
etc.,  the  usual  formula  being 
H  — C02  =  2  :  i. 

Produces  no  indol.  Indol-production  marked. 

Growth   in   milk   unaccompanied  Milk  coagulated. 

by  coagulation. 

Gives  the  Widal  reaction  with  the  Does    not    react    with    typhoid 

serum  of  typhoid  blood.  blood. 

*  "  Amer.  Jour.  Med.  Sci.,"  1895,  110,  p.  287. 
43 


674  Bacillus  Coli 

Colon  Bacillus  in  Drinking  Water. — Much  importance 
attaches  to  the  presence  or  absence  of  colon  bacilli  in  judging 
the  potability  of  drinking  waters. 

It  is  a  speculation  whether  the  colon  bacilli  were  originally 
micro-organisms  of  the  soil  that  accidentally  found  their 
way  into  the  congenial  environment  of  the  intestine  and 
there  took  up  permanent  residence,  or  whether  they  have 
always  been  intestinal  parasites  and  have  been  discharged 
with  the  excrement  of  animals  until  the  soil  has  become 
generally  infected  with  them.  However  this  may  be, 
they  are  at  present  found  in  the  intestinal  canals  of  all 
animals,  and  in  pretty  much  all  soils,  their  number  being 
greatest  in  manured  soils.  From  the  soil  it  is  inevitable 
that  the  organisms  shall  pass  into  the  surface  waters,  which 
with  few  exceptions  will  be  found  to  contain  them.  The 
numbers,  however,  can  be  made  use  of  to  indicate  the  quality 
of  the  water,  a  few  organisms  indicating  that  the  water  is 
pure,  many  that  it  is  freely  mixed  with  surface  washings. 

As  sewage  contains  as  many  as  i  ,000,000  colon  bacilli  per 
cubic  centimeter  and  pure  water  very  often  o  per  cubic 
centimeter  (only  i  cubic  centimeter  being  examined  at  a 
time),  the  number  of  bacilli  per  cubic  centimeter  can  be 
expressed  as  indicating  the  amount  of  sewage  pollution. 
The  number  of  colon  bacilli  in  the  water  is,  therefore,  of 
importance  in  determining  its  potability,  and  in  cases  in 
which  the  quality  of  the  water  is  doubtful,  should  always 
be  employed.  There  is  no  infallible  criterion  for  judging 
the  quality  of  water,  but  most  American  bacteriologists 
are  in  accord  in  concluding  that  when  the  repeated  examina- 
tion of  i  c.c.  samples  shows  the  presence  of  numerous  colon 
bacilli,  the  water  is  seriously  polluted  and  doubtfully  potable, 
but  when  samples  of  i  c.c.  are  without  colon  bacilli  or 
contain  very  few,  the  water  is  safe. 

Another  important  matter  in  regard  to  the  colon  bacillus 
in  water  is  the  presence  or  absence  of  certain  characters 
by  which  one  can  judge  how  recently  it  has  ended  its  intes- 
tinal parasitism  and  taken  up  a  saprophytic  life.  The 
chief  of  these  characters  is  the  ability  to  ferment  lactose. 
Only  recently  isolated  organisms  manifest  this  fermentative 
power  in  the  laboratory,  so  that  when  organisms  capable 
of  fermenting  lactose  are  found,  one  can  suppose  that  they 
result  from  recent  sewage  pollution. 

Many  media  have  been  recommended  for  the  rapid  detec- 


Bacilli  Resembling  the  Typhoid  Bacillus       675 

tion  of  the  colon  bacilli  in  water,  the  favorite  at  the  present 
time  probably  being  the  litmus-lactose-agar  plate  (q.  v.)  of 
Wiirtz.*  This  depends  upon  the  fermentative  and  acid- 
producing  power  of  the  bacillus,  which  is  shown  through 
the  presence  of  red  colonies  (acid  producers)  on  the  else- 
where blue  plate.  These  red  colonies  are  then  fished  up 
and  transplanted  to  appropriate  media  for  further  study. 

Kline  f  substitutes  lactose  for  the  glucose  in  this  medium, 
pointing  out  that  by  so  doing  one  at  once  differentiates 
between  typical  colon  bacilli  which  ferment  lactose  and 
atypical  varieties  which  do  not. 

Other  media  and  methods  useful  in  studying  the  colon 
bacilli  are  also  discussed  in  the  chapter  upon  Typhoid  Fever 
(q.  v.). 

BACILLUS  ENTERITIDIS  (GARTNER). 

General  Characteristics.— A  motile,  flagellated,  non-sporogenous, 
non-chromogenic,  non-liquefying,  aerogenic,  aerobic  and  optionally 
anaerobic,  pathogenic  bacillus  staining  by  the  ordinary  methods,  but 
not  by  Gram's  method. 

This  bacillus  was  first  cultivated  by  A.  Gartner  {  from  the 
flesh  of  a  cow  slaughtered  because  of  an  intestinal  disease, 
and  from  the  spleen  of  a  man  poisoned  by  eating  meat 
obtained  from  it.  The  bacillus  was  subsequently  found  by 
Karlinski  and  Lubarsch  in  other  cases  of  meat-poisoning. 

Morphology. — The  bacillus  closely  resembles  Bacillus 
coli  communis.  It  is  short  and  thick,  is  surrounded  by  a 
slight  capsule,  is  actively  motile,  and  has  flagella. 

Staining. — It  stains  irregularly  with  the  ordinary  solu- 
tions, but  not  by  Gram's  method.  It  has  no  spores. 

Cultivation. — Upon  gelatin  plates  it  forms  round,  pale 
gray,  translucent  colonies.  It  does  not  liquefy  the  gelatin. 
The  deep  colonies  are  brown  and  spheric.  The  growth  on 
agar-agar  is  similar  to  that  of  the  colon  bacillus.  The 
organism  produces  no  indol,  coagulates  milk  in  a  few  days, 
and  reduces  litmus.  Its  fermentative  powers  have  not  been 
sufficiently  studied,  but  is  known  to  ferment  dextrose  media. 
Upon  potato  it  forms  a  yellowish- white,  shining  layer. 

*  "Archiv.  de  med.  Experimental, "  iv,  p.  85,  1892. 
t  "British  Medical  Journal,"  Oct.  27,  1906,  p.  1090. 
J  "Korrespond.  d.  allg.  arztl.  Ver.  von  Thuring,"  1888,  9. 


676  Bacillus  Faecalis 

Pathogenesis. — The  bacillus  is  pathogenic  for  mice, 
guinea-pigs,  pigeons,  lambs,  and  kids,  but  not  for  dogs, 
cats,  rats,  or  sparrows.  The  infection  may  be  fatal  for 
mice  and  guinea-pigs,  whether  given  subcutaneously,  intra- 
peritoneally,  or  by  the  mouth. 

Lesions. — The  bacilli  are  found  scattered  throughout  the 
organs  in  small  groups,  resembling  those  of  the  typhoid 
bacillus. 

At  the  autopsy  a  marked  enteritis  and  swelling  of  the 
lymphatic  follicles  and  patches,  with  occasional  hemor- 
rhages, are  found.  The  bacilli  occur  in  the  intestinal  con- 
tents. The  spleen  is  somewhat  enlarged. 

The  bacillus  is  differentiated  from  the  colon  bacillus 
chiefly  by  the  absence  of  indol-production,  by  its  ability  to 
produce  infection  when  ingested,  and  by  the  fact  that  it 
elaborates  a  toxic  substance  capable  of  producing  symp- 
toms similar  to  those  seen  in  the  infection. 

It  may  be  distinguished  from  Bacillus  lactis  aerogenes 
by  its  motility.  It  closely  resembles  certain  water  bacteria; 
but  its  pathogenesis  can  be  made  use  of  for  assisting  in  its 
differentiation  in  doubtful  cases. 


BACILLUS  F^CALIS  ALKALIGENES  (PETRUSCHKY). 

General  Characteristics.— A  motile,  flagellated,  non-sporogenous, 
non-liquefying,  non-chromogenic,  non-aerogenic,  aerobic  and  option- 
ally anaerobic,  non-pathogenic  bacillus  of  the  intestine,  staining  by 
ordinary  methods,  but  not  by  Gram's  method. 

This  bacillus  has  occasionally  been  isolated  by  Petruschky  * 
and  others  from  feces.  It  closely  resembles  the  typhoid 
bacillus,  being  short,  stout,  with  round  ends,  forming  no 
spores,  staining  with  the  usual  dyes,  but  not  by  Gram's 
method,  being  actively  motile,  and  having  numerous  flagella. 
It  does  not  liquefy  gelatin,  does  not  coagulate  milk,  produce 
gas,  or  form  indol.  Its  pathogenic  powers  for  the  lower  ani- 
mals are  similar  to  those  of  the  typhoid  bacillus. 

It  grows  more  luxuriantly  than  the  typhoid  bacillus  upon 
potato,  producing  a  brown  color,  and  generates  a  strong 
alkali  when  grown  in  litmus- whey.  Its  cultures  are  not 
agglutinated  by  the  typhoid  serums. 

*"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  xix,  187. 


Bacilli  Resembling  the  Typhoid  Bacillus       677 


BACILLUS  PSITTACOSIS  (NOCARD). 

General  Characteristics.— A  motile,  flagellated,  non-sporogenous, 
aerobic,  optionally  anaerobic,  non-chromogenic,  aerogenic,  pathogenic, 
non-liquefying  bacillus,  staining  by  the  ordinary  methods,  but  not 
by  Gram's  method. 

This  micro-organism  was  discovered  by  Nocard,*  who  first 
observed  it  in  1892  in  certain  cases  of  psittacosis,  or  epidemic 
pneumonia,  traceable  to  infection  from  diseased  parrots.  The 
original  paper  contained  an  excellent  account  of  the  specific 
organism. 

The  subsequent  work  of  Gilbert  and  Fournierf  shows  the 
specificity  of  the  micro-organism  to  be  quite  well  established 
and  Nocard's  characterizations  accurate. 

Morphology. — The  bacillus  is  short,  stout,  rounded  at 
the  ends,  and  actively  motile.  It  is  provided  with  flagella, 
but  forms  no  spores.  It  resembles  the  typhoid  and  the 
colon  bacilli  and  is  evidently  a  form  intermediate  between 
the  two. 

Isolation. — Gilbert  and  Fournier  succeeded  in  isolating 
it  from  the  blood  of  a  patient  dead  of  psittacosis,  and  from 
parrots,  by  the  use  of  lactose-litmus  agar.  The  organism 
does  not  alter  the  litmus,  and  if  a  small  percentage  of  carbolic 
acid  be  added  to  the  culture-media,  it  grows  as  does  the 
typhoid  bacillus. 

Cultivation. — The  colonies,  agar-agar  and  gelatin  cul- 
tures, closely  resemble  those  of  the  typhoid  fever  organism. 
Upon  potato  it  more  closely  resembles  the  colon  bacillus. 
Bouillon  becomes  clouded. 

Metabolic  Products. — In  bouillon  containing  sugars  the 
micro-organism  is  found  to  ferment  dextrose,  but  not  lactose. 
Milk  is  not  coagulated  and  not  acidulated.  No  indol  is 
formed. 

Pathogenesis. — Bacillus  psittacosis  can  be  immediately 
differentiated  from  the  typhoid  and  colon  bacilli  by  its 
peculiar  pathogenesis.  It  is  extremely  virulent  for  parrots, 
producing  a  fatal  infection  in  a  short  time.  White  and 
gray  mice  and  pigeons  are  equally  susceptible.  Ten  drops 
of  a  bouillon  culture  injected  in  the  ear-vein  of  a  rabbit 

*"  Seance  du  Conseil  d'hygiene  publique  et  Salubrite  du  Departe- 
ment  de  la  Seine,"  March  24,  1893. 

f  "Comptes  de  la  Societe  de  Biologic,"  1896;  "LaPresse  medicate," 
Jan.  16,  1897. 


678  Bacillus  Suipestifer 

kill  it  in  from  twelve  to  eighteen  hours.  Guinea-pigs  are 
more  resistant.  Subcutaneous  injection  of  dogs  produces  a 
hard,  painful  swelling,  which  persists  for  a  short  time  and 
then  disappears  without  suppuration.  It  is  also  infectious 
for  man,  a  number  of  epidemics  of  peculiar  pneumonia,  char- 
acterized by  the  presence  of  the  bacillus  in  the  blood,  traceable 
to  diseased  parrots,  having  been  reported. 

Differentiation. — Bacillus  psittacosis  can  best  be  differ- 
entiated from  the  typhoid  and  the  colon  bacilli  and  others 
of  the  same  group  by  its  pathogenesis  and  by  the  reaction 
of  agglutination.  Typhoid  immune  serum  produces  some 
small  agglutinations,  but  a  comparison  between  these  and 
the  agglutinations  formed  by  cultures  of  the  typhoid  bacillus 
shows  immediately  that  the  micro-organisms  are  dissimilar. 
Differentiation  is  best  made  out  when  the  prepared  hanging- 
drop  specimens  of  serums  and  cultures  are  kept  for  some 
hours  in  an  incubating  oven.  It  is  not  known  whether  the 
bacillus  is  peculiar  to  the  intestines  of  parrots,  invading  their 
tissues  when  they  become  ill,  or  whether  it  is  a  purely  patho- 
genic micro-organism  found  only  in  psittacosis. 


BACILLUS  SUIPESTIFER  (SALMON  AND  SMITH). 

General  Characteristics.— An  actively  motile,  flagellated,  non- 
sporogenous,  non-chromogenic,  non-liquefying,  aerobic  and  optionally 
anaerobic,  aerogenic  bacillus  pathogenic  for  hogs  and  other  animals. 
It  stains  by  the  ordinary  methods,  but  not  by  Gram's  method.  It 
ferments  dextrose,  lactose,  and  sucrose,  but  does  not  form  indol  or 
coagulate  or  acidulate  milk. 

Hog-cholera,  or  "pig  typhoid,"  as  the  English  call  it,  is  a 
common  epidemic  disease  of  swine,  which  at  times  kills  90 
per  cent,  of  the  infected  animals,  and  thus  causes  immense 
losses  to  breeders.  Salmon  estimates  that  the  annual  losses 
from  this  disease  in  the  United  States  range  from  $10,000,000 
to  $25,000,000.  For  years  it  was  thought  to  be  caused  by  the 
Bacillus  suipestifer,  but  DeSchweinitz  and  Dorset*  were 
able  to  transmit  the  disease  from  one  hog  to  another  in  certain 
of  the  body  fluids  that  had  been  passed  through  the  finest 
porcelain  filters  and  were  shown  by  inoculation  and  cultiva- 
tion to  be  free  of  bacilli.  It  therefore  depends  upon  a  fil- 
terable and  unknown  virus. 

*  "Circular  No.  41  of  Bureau  of  Animal  Industry,"  U.S.  Dept.  of 
Agriculture,  Washington,  D.  C. 


Bacilli  Resembling  the  Typhoid  Bacillus       679 

This  observation  was  received  with  approval  by  those  who 
had  any  experience  with  the  effect  of  hog-cholera  bacilli 
upon  hogs,  all  of  whom  must  have  observed  that  though 
infection  with  the  bacilli  occasionally  caused  the  death  of  an 
animal,  the  dead  animal  usually  did  not  show  the  typical 
lesions  of  the  disease  and  never  infected  other  animals 
with  which  it  was  kept.  The  papers  upon  the  subject  by 
Dorset,  Bolton,  and  McBryde*  and  by  Dorset,  McBryde,  and 
Nilesf  are  worth  reading. 

These  investigations  entirely  change  our  ideas  of  the  im- 
portance of  the  hog-cholera  bacillus,  whose  relation  to  the 
disease  now  comes  to  resemble  that  of  Bacillus  icteroides 
to  yellow  fever. 

The  bacillus  of  hog-cholera  was  first  found  by  Salmon  and 
Smith,|  but  was  for  a  long  time  confused  with  the  bacillus 
of  "swine-plague,"  which  it  closely  resembles,  and  in  associa- 
tion with  which  it  frequently  occurs.  It  is  a  member  of 
the  group  of  bacteria  to  which  Bacillus  icteroides  and  B. 
typhi  murium  belong.  The  organism  was  secured  by 
Smith  from  the  spleens  of  more  than  500  hogs.  It  occurs 
in  the  blood  and  in  all  the  organs,  and  has  also  been  culti- 
vated from  the  urine. 

Morphology. — The  organisms  appear  as  short  rods  with 
rounded  ends,  1.2  to  1.5  ^  long  and  0.6  to  0.7  ^  in  breadth. 
They  are  actively  motile  and  possess  longflagella  (peritrichia) , 
easily  demonstrable  by  the  usual  methods  of  staining.  No 
spore  production  has  been  observed.  In  general  the  bacillus 
resembles  that  of  typhoid  fever.  It  stains  readily  by  the 
ordinary  methods,  but  not  by  Gram's  method. 

Cultivation. — No  trouble  is  experienced  in  cultivating 
the  bacilli,  which  grow  well  in  all  the  media  under  aerobic 
and  anaerobic  conditions. 

Colonies. — Upon  gelatin  plates  the  colonies  become 
visible  in  from  twenty-four  to  forty-eight  hours,  the  deeper 
ones  appearing  spheric  with  sharply  defined  borders.  The 
surfaces  are  brown  by  reflected  light,  and  without  markings. 

*  "Bull.  No.  72  of  Bureau  of  Animal  Industry,"  U.  S.  Dept.  Agricul- 
ture, Washington,  D.  C.,  1905. 

t  "Bull.  No.  102  of  Bureau  of  Animal  Industry,"  U.  S.  Dept.  Agri- 
culture, Washington,  D.  C.,  Jan.  18,  1908. 

t" Reports  of  the  Bureau  of  Animal  Industry,"  1885-91;  and 
"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Bd.  ix,  Nos.  8,  9,  and  10,  March 
2,  1891. 


68o  Bacillus  Suipestifer 

They  are  rarely  larger  than  0.5  mm.  in  diameter  and  are 
homogeneous  throughout.  The  superficial  colonies  have 
little  tendency  to  spread  upon  the  gelatin.  They  rarely 
reach  a  greater  diameter  than  2  mm.  The  gelatin  is  not 
liquefied. 

Upon  agar-agar  they  attain  a  diameter  of  4  mm.  and 
have  a  gray,  translucent  appearance  with  polished  surface. 
They  are  round  and  slightly  arched. 

Gelatin. — In  gelatin  punctures  the  growth  takes  the  form 
of  a  nail  with  a  flat  head.  There  is  nothing  characteristic 
about  it.  The  medium  is  not  liquefied. 

Agar-agar. — Linear  cultures  upon  agar-agar  present  a 
translucent,  circumscribed,  grayish,  smeary  layer  without 
characteristic  appearances. 

Potato. — Upon  potato  a  yellowish  coating  is  formed, 
especially  when  the  culture  is  kept  in  the  thermostat. 

Bouillon. — Bouillon  made  with  or  without  peptone  is 
clouded  in  twenty-four  hours.  When  the  culture  is  allowed 
to  stand  for  a  couple  of  weeks  without  being  disturbed,  a 
thin  surface  growth  can  be  observed. 

Milk  is  an  excellent  culture-medium,  but  is  not  visibly 
changed  by  the  growth  of  these  bacteria.  Its  reaction  re- 
mains alkaline. 

Vital  Resistance. — The  bacillus  is  hardy.  Smith  found 
it  vital  after  being  kept  dry  for  four  months.  It  ordinarily 
dies  sooner,  however,  and  I  have  experienced  difficulty  in 
keeping  it  in  the  laboratory  for  any  length  of  time  unless 
frequently  transplanted.  The  thermal  death-point  is  54°  C., 
maintained  for  sixty  minutes. 

Metabolic  Products. — Gas  Production. — The  hog-chol- 
era bacillus  is  a  copious  gas-producer,  capable  of  break- 
ing up  dextrose  and  lactose  into  CO2,  H,  and  an  acid,  which, 
formed  late,  eventually  checks  its  further  development.  It 
does  not  ferment  saccharose. 

Indol. — No  indol  and  no  phenol  are  formed  in  the  culture- 
media. 

Toxin. — In  pure  cultures  of  the  hog-cholera  bacillus 
Novy*  found  a  poisonous  base  with  the  probable  composition 
C16H26N2,  which  he  gave  the  provisional  name  "susotoxin." 
In  doses  of  100  mg.  the  hydrochlorid  of  this  base  causes  con- 
vulsive tremors  and  death  within  one  and  one-half  hours  in 

*  "Medical  News,"  1900,  p.  231. 


Bacilli  Resembling  the  Typhoid  Bacillus       68 1 

white  rats.  He  has  also  obtained  a  poisonous  protein  of 
which  50  mg.  were  fatal  for  white  rats,  and  which  immu- 
nized them  against  highly  virulent  hog-cholera  organisms 
when  administered  by  repeated  subcutaneous  injection. 

De  Schweinitz*  has  also  separated  a  slightly  poisonous 
base  which  he  calls  "sucholotoxin,"  and  a  poisonous  protein 
that  crystallizes  in  white,  translucent  plates  when  dried  over 
sulphuric  acid  in  vacua,  forms  needle-like  crystals  with  pla- 
tinic  chlorid,  and  was  classed  among  the  albumoses. 

Pathogenesis. — The  bacillus  is  disappointing  in  its 
effects  upon  hogs.  When  it  is  subcutaneously  or  intra- 
venously introduced  into  such  animals  or  fed  to  them,  they 
sometimes  show  no  signs  of  disease;  sometimes  show  fever 
and  depression,  but  rarely  sicken  enough  to  die.  Animals 
thus  made  ill  do  not  communicate  the  disease  to  others. 

Smith  found  that  0.75  c.c.  of  a  bouillon  culture  injected 
into  the  breast  muscles  of  pigeons  would  kill  them. 

In  Smith's  experiments  one  four-millionth  of  a  cubic 
centimeter  of  a  bouillon  culture  injected  subcutaneously 
into  a  rabbit  was  sufficient  to  cause  its  death.  The  tem- 
perature abruptly  rises  2°  to  3°  C.,  and  remains  high  until 
death.  Subcutaneous  injection  of  larger  quantities  may 
kill  in  five  days.  Injected  intravenously  in  small  doses  the 
bacillus  may  kill  rabbits  in  forty-eight  hours. 

Agglutination. — Pitfieldf  found  that  after  a  single  in- 
jection of  a  killed  bouillon  culture  of  the  bacillus  into  a 
horse,  the  serum,  which  originally  had  very  slight  agglutina- 
tive power,  showed  a  decided  increase.  If  the  horse  be 
immunized  to  large  doses  of  such  sterile  cultures,  the  serum 
becomes  so  active  that  with  a  dilution  of  i  :  10,000  a  typical 
agglutination  occurs  in  sixty  minutes. 

McClintock,  Boxmeyer  and  SifferJ  found  that  the  serum 
of  normal  hogs  agglutinates  strains  of  ordinary  hog-cholera 
bacilli  in  dilutions  occasionally  as  high  as  i  :  250,  and  con- 
sider reaction  in  a  dilution  of  less  than  i  :  300  without 
diagnostic  value. 

"Medical  News,"  1900,  p.  237. 
f  "Microscopical  Bulletin,"  1897,  p.  35. 
J  "Jour,  of  Infectious  Diseases,"  March  i,  1905,  vol.  n,  No.  2,  p.  351. 


682  Bacillus  Icteroides 

BACILLUS  ICTEROIDES  (SANARELU). 

General  Characteristics.— An  actively  motile,  flagellated,  non- 
sporogenous,  non-liquefying,  non-chromogenic,  aerogenic,  aerobic  and 
optionally  anaerobic,  pathogenic  bacillus  which  stains  by  the  ordinary 
method,  but  not  by  Gram's  method.  It  produces  indol,  but  does  not 
coagulate  milk. 

Sanarelli  regarded  this  bacillus  as  the  specific  organism  of 
yellow  fever.  He  found  it  in  n  autopsies  upon  yellow 
fever  cases,  but  always  in  association  with  streptococci, 
colon  bacilli,  proteus,  and  other  organisms.  It  is  found  in 
the  blood  and  tissues,  and  not  in  the  gastro-intestinal  tract, 
and  isolation  of  the  organism  was  possible  in  only  58  per 
cent,  of  the  cases,  and  only  in  rare  instances  was  accom- 
plished during  life. 

Distribution. — By  suitable  methods  it  can  be  found  in 
the  organs  of  yellow  fever  cadavers,  usually  aggregated  in 
small  groups,  in  the  capillaries  of  the  liver,  kidneys,  and 
other  organs.  The  best  method  of  demonstration  is  to 
keep  a  fragment  of  liver,  obtained  from  a  body  soon  after 
death,  in  the  incubator  at  37°  C.  for  twelve  hours,  and 
allow  the  bacteria  to  multiply  in  the  tissue  before  exam- 
ination. 

Morphology. — The  bacillus  presents  nothing  morpho- 
logically characteristic.  It  is  a  small  pleomorphous  bacillus 
with  rounded  ends,  usually  joined  in  pairs.  It  is  2  104  ^  in 
length,  and,  as  a  rule,  two  or  three  times  longer  than  broad. 
It  is  actively  motile  and  has  flagella.  It  does  not  form  spores. 

Staining. — It  stains  by  the  usual  methods,  but  not  by 
Gram's  method. 

Cultivation. — The  bacillus  can  be  grown  upon  the  usual 
media.  It  grows  readily  at  ordinary  room  temperatures, 
but  best  at  37°  C. 

Colonies. — Upon  gelatin  plates  it  forms  rounded,  trans- 
parent, granular  colonies,  which  during  the  first  three  or 
four  days  somewhat  resemble  leukocytes.  The  granular 
appearance  becomes  continuously  more  marked,  and  usu- 
ally an  opaque  central  or  peripheral  nucleus  is  seen.  In 
time  the  entire  colony  becomes  opaque,  but  does  not  liquefy 
gelatin. 

Gelatin. — Stroke  cultures  on  obliquely  solidified  gelatin 
show  brilliant,  opaque,  white  colonies  resembling  drops  of 
milk.  The  medium  is  not  liquefied. 

Bouillon. — In  bouillon  it  develops  slowly,  without  either 
pellicle  or  flocculi. 


Bacilli  Resembling  the  Typhoid  Bacillus       683 


Agar-agar. — The  culture  upon  agar-agar  is  said  to  be 
characteristic. 

The  peculiar  and  characteristic  appearances  of  the  colonies 
do  not  develop  if  grown  at  37°  C.;  but  at  20°  to  22°  C.  the 
colonies  appear  rounded,  whitish,  opaque,  and  prominent, 
like  drops  of  milk.  This  appearance  of 
the  colonies  also  shows  well  if  the  cul- 
tures are  kept  for  the  first  twelve  to  six- 
teen hours  at  37°  C.,  and  afterward  at 
the  room  temperature,  when  the  colonies 
will  show  a  flat  central  nucleus,  trans- 
parent and  bluish,  surrounded  by  a 
prominent  and  opaque  zone,  the  whole 
resembling  a  drop  of  sealing-wax.  Sana- 
relli  refers  to  this  appearance  as  consti- 
tuting the  chief  diagnostic  feature  of 
Bacillus  icteroides.  It  can  be  observed 
in  twenty-four  hours. 

Blood-serum. — Upon  blood-serum  the 
growth  is  very  meager. 

Potato. — The  growth  upon  potato  cor- 
responds with  that  of  the  bacillus  of 
typhoid  fever. 

Vital  Resistance. — It  strongly  resists 
drying,  but  dies  when  exposed  in  cultures 
to  a  temperature  of  60°  C.  for  a  few 
minutes,  and  is  killed  in  seven  hours  by 
the  solar  rays.  It  can  live  for  a  consider- 
able time  in  sea- water. 

Metabolism. — The  bacillus  is  an  op- 
tional anaerobe.  It  slowly  ferments  dex- 
trose, lactose,  and  saccharose,  forming 
gas  only  in  dextrose  solutions  in  which 
there  are  no  other  sugars.  It  does  not 
coagulate  milk.  In  the  cultures  a  small 
amount  of  indol  is  formed. 

Pathogen esis. — The  bacillus  is  patho- 
genic for  the  domestic  animals,  all  mammals  seeming  to  be 
more  or  less  sensitive  to  it.  Birds  are  often  immune. 
White  mice  are  killed  in  five  days,  guinea-pigs  in  from  eight 
to  twelve  days,  rabbits  in  from  four  to  five  days,  by  virulent 
cultures.  The  morbid  changes  present  include  splenic  tumor, 
hypertrophy  of  the  thymus,  and  adenitis.  In  the  rabbit 


Fig.  229. — Cul- 
ture of  bacillus 
icteroides  on  agar 
(Sanarelli). 


684  Bacillus  Murium 

there    are,   in    addition,    nephritis,    enteritis,    albuminuria, 
hemoglobinuria,  and  hemorrhages  into  the  body  cavities. 

Sanarelli  states  that  the  dog  is  the  most  susceptible  animal. 
When  this  animal  is  injected  intravenously,  symptoms  ap- 
pear almost  immediately  and  recall  the  clinical  and  anatomic 
features  of  yellow  fever  in  man.  The  most  prominent  symp- 
tom in  the  dog  is  vomiting,  which  begins  directly  after  the 
penetration  of  the  virus  into  the  blood,  and  continues  for  a 
long  time.  Hemorrhages  appear  after  the  vomiting,  the 
urine  is  scanty  and  albuminous,  or  is  suppressed  shortly 
before  death.  Grave  jaundice  was  once  observed. 


BACILLUS  TYPHI  MURIUM  (Z,OFF%ER). 

General  Characteristics. — A  motile,  flagellated,  non-sporogenous, 
non-liquefying,  non-chromogenic,  non-aerogenic,  aerobic  and  optionally 
anaerobic  bacillus,  pathogenic  for  mice  and  other  small  animals,  stain- 
ing by  the  ordinary  methods,  but  not  by  Gram's  method.  It  acidulates 
but  does  not  coagulate  milk. 

Bacillus  typhi  murium  was  discovered  by  Loffler*  in 
1889,  when  it  created  havoc  among  the  mice  in  his  labora- 
tory at  Greifswald. 

Morphology. — The  organism  bears  a  close  resemblance 
to  that  of  typhoid  fever,  sometimes  appearing  short,  some- 
times long  and  flexible.  There  are  many  long  and  curly 
flagella  with  peritrichial  arrangement,  and  the  organism  is 
actively  motile.  It  does  not  produce  spores. 

Staining. — It  stains  with  the  ordinary  dyes,  but  rather 
better  with  Loffler's  alkaline  methylene-blue,  not  by  Gram's 
method. 

Isolation. — The  bacilli  were  first  isolated  from  the  blood 
of  dead  mice. 

Cultivation. — Their  cultivation  presents  no  difficulties. 

Colonies. — Upon  gelatin  plates  the  deep  colonies  are  at 
first  round,  slightly  granular,  transparent,  and  grayish. 
Later  they  become  yellowish  brown  and  granular.  Super- 
ficial colonies  are  similar  to  those  of  the  typhoid  bacillus. 

Gelatin. — In  gelatin  punctures  there  is  no  liquefaction. 
The  growth  takes  place  principally  upon  the  surface,  where 
a  grayish-white  mass  slowly  forms,  and  together  with  the 
growth  in  the  puncture  suggests  a  large  flat-headed  nail. 

Agar-agar. — Upon  agar-agar  a  grayish-white  growth  de- 
void of  peculiarities  occurs. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  xi,  p.  129. 


Bacilli  Resembling  the  Typhoid  Bacillus       685 

Potato. — Upon  potato  a  rather  thin  whitish  growth  may 
be  observed  after  a  few  days. 

Milk. — The  bacillus  grows  well  in  milk,  causing  acid  reac- 
tion, without  coagulation. 

Bouillon. — In  bouillon  it  produces  clouding.  There  is  no 
fermentation  of  saccharose,  dextrose,  lactose,  or  levulose. 

Pathogenesis. — The  organism  is  pathogenic  for  mice  of 
all  kinds,  which  succumb  in  from  one  to  two  days  when 
inoculated  subcutaneously,  and  in  from  eight  to  twelve 
days  when  fed  upon  material  containing  the  bacillus.  The 


Fig.  230. — Bacillus  typhi  murium  (Migula). 


bacilli  multiply  rapidly  in  the  blood-  and  lymph-channels, 
and  cause  death  from  septicemia. 

Loffler  expressed  the  opinion  that  this  bacillus  might  be 
of  use  in  ridding  infested  premises  of  mice,  and  its  use  for 
this  purpose  has  been  satisfactory  in  many  places.  He  has 
succeeded  in  ridding  fields  so  infested  with  mice  as  to  be 
useless  for  agricultural  purposes,  by  saturating  bread  with 
bouillon  cultures  of  the  bacillus  and  distributing  it  near  their 
holes.  The  bacilli  not  only  killed  the  mice  that  had  eaten 
the  bread,  but  also  infected  others  which  ate  their  dead 
bodies,  the  extermination  progressing  until  scarcely  a  mouse 
remained  in  the  field. 

In  discussing  the  practical  employment  of  this  bacillus 


686  Bacillus  Murium 

for  the  satisfactory  destruction  of  field-mice,  Brunner*  calls 
attention  to  certain  conditions  that  are  requisite:  (i)  It  is 
necessary,  first  of  all,  to  attack  extensive  areas  of  the  in- 
vaded territory,  and  not  to  attempt  to  destroy  the  mice  of  a 
small  field  into  which  an  indefinite  number  of  fresh  animals 
may  immediately  come  from  surrounding  fields.  The 
country  people,  who  are  the  sufferers,  should  combine  their 
efforts  so  as  to  extend  the  benefits  widely.  (2)  The  prepa- 
ration of  the  cultures  is  a  matter  of  importance.  Agar-agar 
cultures  are  most  readily  transportable.  They  are  broken  up 
in  water,  well  stirred,  and  the  liquid  poured  upon  a  large 
number  of  small  pieces  of  broken  bread.  These  are  then 
distributed  over  the  ground  with  care,  being  dropped  into 
the  fresh  mouse-holes,  and  pushed  sufficiently  far  in  to 
escape  the  effects  of  sunlight  upon  the  bacilli.  Attention 
should  be  paid  to  holes  in  walls,  under  railway  tracks,  etc., 
and  other  places  where  mice  live  in  greater  freedom  from 
disturbance  than  in  the  fields.  (3)  The  destruction  of 
the  mice  should  be  attempted  only  at  a  time  of  the  year 
when  their  natural  food  is  not  plenty.  By  observing  these 
precautions  the  mice  can  be  eradicated  in  from  eight  to 
twelve  days.  In  the  course  of  two  years  no  less  than  250,000 
cultures  were  distributed  from  the  Bacteriological  Labora- 
tory of  the  Tierarznei  Institut  in  Vienna,  for  the  purpose  of 
destroying  field-mice. 

The  bacilli  are  not  pathogenic  for  animals,  such  as  the  fox, 
weasel,  ferret,  etc.,  that  feed  upon  the  mice,  do  not  affect 
man  in  any  way,  and  so  seem  to  occupy  a  useful  place  in 
agriculture  by  destroying  the  little  but  almost  invincible 
enemies  of  the  grain. 

A  similar  organism,  secured  from  an  epidemic  among 
field-mice  and  greatly  increased  in  virulence  by  artificial 
manipulation,  has  been  recommended  by  Danyszf  for  the 
destruction  of  rats.  When  subjected  to  a  thorough  study 
by  RosenauJ  this  organism  was  found  to  be  identical  with 
Bacillus  typhi  murium.  It  is,  however,  too  uncertain  in 
action  to  be  relied  upon  for  the  destruction  of  rats  in  plague- 
threatened  cities  for  which  it  was  suggested. 

*  "Centralbl.  f.  Bakt.,"  etc.,  Jan.  19,  1898,  Bd.  xxm,  No.  2,  p.  68. 
t  "Ann.  de  1'Inst.  Pasteur,"  April,  1900. 

I  "Bulletin  No.  5  of  the  Hygienic  Laboratory  of  the  U.S.  Marine 
Hospital  Service,"  Washington  D.  C.,  1901. 


CHAPTER  XXVI. 

DYSENTERY. 

DYSENTERY  is  an  acute,  subacute,  or  chronic,  infectious  coli- 
tis, usually  characterized  by  an  acute  onset,  mild  fever, 
pain  in  the  abdomen,  rectal  tenesmus,  and  the  passage  of 
frequent,  usually  small,  mucous  and  bloody  evacuations 
from  the  rectum. 

The  disease  was  known  to  the  ancients.  It  was  probably 
dysentery  that  is  meant  by  "emerods"  in  describing  an 
epidemic  that  took  place  among  the  people  of  Israel  during 
the  time  of  the  Judges.  Hippocrates  differentiated  between 
diarrhea  and  dysentery. 

Sporadic  cases  of  the  disease  occur  in  almost  all  countries, 
the  number  of  such  increasing  as  the  equator  is  approached. 
In  addition  to  these  sporadic  cases  epidemics  not  infrequently 
appear.  Though  such  may  break  out  at  any  time  in  towns 
or  cities,  they  are  more  apt  to  occur  when  unusual  activities 
of  the  people  are  in  progress.  The  most  frequent  of  these 
is  military,  and  armies  are  apt  to  be  the  greatest  sufferers. 
The  incidence  of  dysentery  in  the  Federal  Army  during  the 
War  of  the  Rebellion  was  appalling.  Woodward*  states 
that  there  were  259,071  cases  of  acute  and  28,451  of  chronic 
dysentery. 

Endemics  also  occur  from  time  to  time  and  assume  devas- 
tating proportions,  as  in  Japan,  where  between  1878  and  1899 
there  were  1,136,096  cases,  with  275,308  deaths — a  mortality 
of  25.23  per  cent.f  Osier  quotes  Macgregor  as  saying,  "In 
the  tropics  dysentery  is  a  destructive  giant  compared  to 
which  strong  drink  is  a  mere  phantom.  It  is  one  of  the 
great  camp  diseases  and  has  been  more  destructive  to  armies 
than  powder  and  shot." 

The  disease  early  came  under  the  observation  of  the  bac- 
teriologists, and  Klebs,  Ziegler,  Ogata,  Grigorieff,  de  Silvestri, 

*  "Medical  and  Surgical  History  of  the  War  of  the  Rebellion,"  Medi- 
cal, n. 

t  "Public  Health  Reports,"  Jan.  5,  1900,  xv,  No.  i. 

687 


688  Dysentery 

Maggiora,  Arnaud,  Celli  and  Fiocca,  Galli-Valerio,  Vala- 
gussa,  Deycke,  and  others  published  descriptions  of  various 
micro-organisms  isolated  from  dysenteric  stools,  and  looked 
upon  by  their  discoverers  as  the  cause  of  the  disease.  The 
results  were,  however,  so  discordant  that  none  of  the  described 
micro-organisms  could  be  agreed  upon  as  the  excitant  of  the 
disease. 

In  1 860  Lambl*  published  a  description  of  an  ameba  found 
in  the  human  intestine.  No  one  seemed  inclined  to  believe 
that  it  might  have  any  significance  until  much  later. 

In  1875  Loschf  described  an  ameba  which  he  found  in 
great  numbers  in  the  colon  of  a  case  of  dysentery  occurring 
in  St.  Petersburg.  Not  much  notice  was  taken  of  his  paper 
or  much  made  of  his  observation  until  eight  years  later,  when 
Koch  and  Gaffky,  {  in  studying  the  cholera  in  Egypt,  also 
observed  amebas  in  the  intestinal  discharges  in  certain  cases, 
and  Kartulis§  wrote  upon  the  "Etiology  of  the  Dysentery  in 
Egypt,"  which  he  referred  to  these  amebas.  In  America  the 
study  of  these  amebas  was  quickly  taken  up.  Osier  ||  discov- 
ered the  organisms  in  the  evacuations  of  a  case  of  dysentery 
contracted  by  a  patient  during  a  visit  to  Panama.  Council- 
man and  Lafleur**  wrote  a  fine  monograph  upon  "Amebic 
Dysentery,"  while  Quincke  and  Roosff  and  Kruse  and  Pas- 
qualeJt  confirmed  the  observations  and  results  in  Europe. 

Thus  it  came  to  be  recognized  that  an  ameba  was  the 
cause  of  dysentery.  It  was  soon  pointed  out,  however,  that 
there  were  cases  of  dysentery  in  which  no  ameba  could  be 
found  in  the  intestinal  discharges,  or  in  which  they  were  so 
few  that  it  seemed  impossible  that  they  could  be  the  cause  of 
the  disease.  This  was  particularly  impressive  throughout 
the  years  of  the  endemic  dysentery  in  Japan,  already  referred 
to.  Great  numbers  of  cases  occurred,  great  numbers  of 
people  died,  no  amebas  were  found  to  account  for  the  disease. 
It  therefore  occurred  to  Kitasato  that  some  other  causal  agent 
must  be  looked  for,  and  Shiga  took  up  the  problem,  which  was 
*  "Aus.  d.  Franz  Joseph  Kinderspital  zur  Prague,"  1860,  I,  326. 
f'Virchow's  Archives,"  1875,  Bd.  LXV. 

t  "Behricht  iiber  die  Erforschung  du  Cholera,"  1883;  "Arbeiten  aus 
d.  Kaiserl.  Gesundheitsamt.,"  in,  65. 
§  "Virchow's  Archives,"  1886,  cv. 
||  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1890,  vu,  736. 

**  "Johns  Hopkins  Hospital  Reports,"  1891,  n. 

ft  "Berliner  klin.  Wochenschrift,"  1893, 

it  "  Zeitschrift  f.  Hygiene,"  etc.,  1894,  xvi. 


Amebic  Dysentery  689 

a  difficult  one,  and  might  not  have  been  solved  had  he  not 
made  use  of  a  new  means  of  investigation,  viz.,  the  phenom- 
enon of  agglutination.  By  studying  such  bacteria  as  could 
be  cultivated  from  the  intestinal  discharges,  with  particular 
reference  to  the  effect  of  the  blood  of  dysenteric  patient's 
blood  in  agglutinating  them,  Shiga  *  succeeded  in  discovering 
a  new  micro-organism  which  he  called  Bacillus  dysenteriae. 
Two  years  afterward  Krusef  investigated  an  outbreak  of 
dysentery  in  an  industrial  section  of  Westphalia  and  found 
the  same  bacillus,  and  FlexnerJ  showed  the  same  bacillus  to 
be  present  in  the  epidemic  dysentery  of  the  Philippine 
Islands. 

Thus  the  discovery  of  Shiga  became  thoroughly  substanti- 
ated, and  it  became  evident  that  in  addition  to  theameba  there 
was  a  bacillus  to  be  reckoned  with  in  the  etiology  of  dysen- 
tery. It  soon  became  evident  that  there  are  two  forms  of 
dysentery,  one  amebic,  the  other  bacillary.  Both  occur  spo- 
radically and  endemically  in  the  tropics  and  in  temperate 
climates,  and  both  may  occur  epidemically,  though  of  the 
two  the  bacillary  form  is  much  more  liable  to  do  so.  Of  the 
chronic  cases  of  dysentery  90  per  cent,  are  amebic. 


L    AMEBIC  DYSENTERY. 

AMOEBA  COLI  (Loscn,  1875);  AMCEBA  DYSENTERIC  (COUN- 
CILMAN AND  LAFLEUR,  1893);  ENTAMCEBA  HisToivYTicA 

(SCHAUDINN,   1903.) 

As  has  been  shown,  amebas  were  first  found  in  the  human 
intestine  by  Lambl;  in  dysentery,  by  Losch,  Koch,  Gaffky, 
Kartulis,  Osier,  Councilman  and  Lafleur,  and  many  others. 
The  welcome  finally  accorded  to  the  organisms  as  excitants 
of  dysentery  was  sufficiently  enthusiastic  to  compensate  for 
the  neglect  of  a  quarter  of  a  century.  In  all  countries  intes- 
tinal ameba  was  looked  for,  and  while  it  was  becoming  clear 
in  the  minds  of  many  pathologists  that  there  could  be  no 
doubt  about  the  role  played  by  the  ameba  in  dysentery,  it 
was  becoming  equally  clear  to  others  that  amebas  were  not 
infrequently  present  in  intestines  in  which  there  was  not  and 
had  not  been  dysenteric  infection,  and  many  cases  of  dysentery 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1898,  xxiv,  817. 
t  "Deutsche  med.  Wochenschrift,"  1900,  No.  40. 
t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1900,  xxvm,  No.  19. 
44 


6go 


Dysentery 


in  which  there  were  no  ameba.  The  lapse  of  time  and  the 
thoroughness  of  investigation  that  came  with  it  solved  both 
of  the  difficulties,  for  it  was  shown  that  there  were  several 
varieties  of  intestinal  amebas  and  also  that  there  was  a 
bacillary  as  well  as  an  amebic  form  of  dysentery. 

Celli  and  Fiocca*  were  the  first  to  study  the  amebas  sys- 
tematically and  to  cultivate  them  upon  artificial  media. 
Councilman  and  Lafleur  pointed  out  that  there  were  two 
varieties  of  amebas  which  they  called  Amoeba  coli  and  Amoeba 
dysenteriae.  The  former  was  supposed  to  be  a  harmless 
commensal,  the  latter  a  pathogenic  organism  and  the  cause 
of  dysentery.  As,  however,  Losch  had  called  the  organism 


Fig.  231. — Amoeba  coli  in  intestinal  mucus,  with  blood-corpuscles  and 
bacteria  (Losch). 

found  in  dysentery  the  Amoeba  coli,  Stiles  declared  the  nomen- 
clature faulty,  and  pointed  out  that  Amoeba  coli,  variety  dys- 
enteriae,  must  be  the  name  of  the  pathogenic  form.  Schau- 
dinn f  reviewed  the  subject  and  grouped  all  of  the  intestinal 
amebas  under  the  following: 

I.  Chlamydophrys  stercorea  (Cienkowsky). 

II.  Amoeba  coli  rhizopodia. 

1.  Entamoeba  coli  (Losch)  (Schaudinn). 

2.  Entamoeba  histolytica  (Schaudinn). 
To  these  has  been  since  added  in  1907: 

Entamoeba  tetragena  (Viereck). 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1894,  xv,  470. 

t  "Arbeiten  aus  d.  Kaiserl.  Gesundheitsamt.,"  1903,  xix,  No.  3. 


Amebic  Dysentery  691 

1.  Entamoeba  Coli  (Losch,  1875). — This  organism  seems 
to  be  a  harmless  commensal,  living  in  the  intestines  of  man, 
many  domestic,  and  many  wild  animals.    It  may  be  abundant 
when  the  reaction  of  the  intestinal  contents  is  neutral  or  al- 
kaline.    It  usually  measures  between  10  and  20  ^  in  diameter 
when  free,  but  when  encysted  from  15  to  50  p.     It  is  sphe- 
roidal when  not  in  motion,  and  under  these  conditions  it  is 
difficult   to   differentiate   endoplasm   and   ectoplasm.     The 
ameboid  movement  is  sluggish  and  the  pseudopods  are  rather 
short,  broad,  and  blunt.     As  they  are  protruded  the  clear 
ectoplasm  becomes  visible.     The   organism  has  a  grayish 
color,  a  finely  granular  cytoplasm,  and  usually  only  a  single 
vacuole.     The  nucleus   is   usually  fairly  well   defined   and 
spherical,  and,  in  addition  to  the  chromatin,  contains  several 
nucleoli.      When  stained  with  polychrome  methylene-blue 
the  ectoplasm  stains  blue;  the  endoplasm,  violet;  and  the 
nucleus,  red. 

Reproduction  usually  takes  place  by  simple  division,  but 
a  form  of  autogamous  sporulation  also  takes  place,  the  organ- 
ism first  becoming  encysted,  the  nucleus  dividing  into  eight 
segments,  and  the  whole  process  eventuating  in  the  formation 
of  eight  young  organisms. 

This  ameba  is  easily  cultivated  upon  artificial  media 
according  to  methods  to  be  described  below. 

It  is  not  pathogenic,  and  all  attempts  to  make  it  damage  the 
the  intestine  of  experiment  animals  have  failed. 

2.  Entamoeba   Histolytica    (Schaudinn). — This   is   now 
recognized  as  the  organism  seen  by  Losch,  Koch,  Kartulis, 
Councilman  and  Lafleur,  and  accepted  as  the  cause  of  the 
amebic  form  of  dysentery.     It  is  found  in  all  parts  of  the 
world,  but  more  frequently  in  tropical  than  colder  climates, 
and  is  present  only  in  the  intestines  of  those  suffering  from 
dysentery.     When  present  it  is  usually  in  great  numbers,  so 
that  its  discovery  in  the  evacuations  is  usually  easy. 

Morphology. — It  is  usually  considerably  larger  than  Enta- 
moeba coli  and  varies  in  diameter  up  to  50  /w.  When  at  rest 
it  is  spherical,  when  active  it  is  very  irregular.  Its  move- 
ment is  active  and  the  pseudopodia  are  larger  and  more 
numerous  than  in  the  other  species.  The  differentiation  of 
ectoplasm  and  endoplasm  is  usually  distinct.  The  former  is 
hyaline,  the  latter  granular.  The  protoplasm  has  a  greenish 
or  yellowish  color.  The  nucleus  is  small,  not  very  distinct. 
There  are  numerous  vacuoles.  When  examined  in  the  in- 


69  2  Dysentery 

testinal  evacuations  of  dysentery  the  protoplasm  commonly 
contains  many  red  blood-corpuscles,  upon  which  the  organ- 
ism seems  to  feed. 

Staining. — When  stained  with  polychrome  methylene-blue 
the  ectoplasm  stains  more  deeply  than  the  endoplasm.  The 
nucleus  contains  relatively  little  chromatin. 

Reproduction. — Multiplication  takes  place  by  binary  divis- 
ion after  karyokinesis  and  by  encystment  and  sporulation. 
The  sporulation  is  quite  different  from  that  seen  in  Enta- 
moeba  coli,  and  only  takes  place  when  conditions  are  unfavor- 
able to  continued  division.  It  is  accomplished  by  a  peculiar 
nuclear  budding,  by  which  chromatin  granules  or  chronidia 
are  pushed  out  from  the  nucleus  toward  the  ectoplasm,  where 
they  develop  into  new  nuclei,  about  which  the  cytoplasm 
collects  until  a  distinct  bud  is  formed  and  cast  off  as  a  small 
but  distinct  new  organism — a  spore  or  bud.  These  when 
separated  are  round  or  oval,  measure  3  to  6  p  in  diameter, 
and  are  surrounded  by  a  yellowish  envelope,  which  resists 
drying  and  the  penetration  of  stains  and  chemicals. 

Craig  gives  (see  page  693)  a  tabulation  of  the  differential 
features  of  Bntamoeba  coli,  Bntamoeba  hystolytica,  and 
Entamoeba  tetragena. 

3.  Entamceba  Tetragena. — This  organism  resembles  En- 
tamoeba hystolytica  more  than  Amoeba  coli,  but  differs  from 
it  in  the  mode  of  reproduction.  The  sporocysts  contain  but 
four  instead  of  eight  spores. 

Isolation  and  Cultivation. — Many  experimenters  have  made 
more  or  less  successful  attempts  to  cultivate  ameba.  Mus- 
grave  and  Clegg,*  whose  interesting  paper  the  student  will 
do  well  to  read,  and  in  which  he  will  find  a  complete  review  of 
all  antecedent  work,  were  able  to  cultivate  a  considerable 
variety  of  amebas  upon  agar-agar  made  of : 

Agar 20. 

Sodium  chlorid 0.3-0.5 

Extract  of  beef 0.3-0.5 

Water 1000.0 

Prepare  as  ordinary  culture  agar,  and  render  i  per  cent, 
alkaline  to  phenolphthalein.  The  finished  medium  is  poured 
into  Petri  dishes.  To  obtain  the  greatest  number  of  most 
active  amebas  the  patient  should  be  given  a  dose  of  a  saline 
purgative,  and  the  fluid  evacuation  resulting  from  its  action 
employed  for  inoculating  the  media.  The  cultures  are,  natu- 

*  "Department  of  the  Interior,  Bureau  of  Government  Laboratories, 
Biological  Laboratory,"  Manila,  Oct.,  1904,  No.  8. 


Amebic  Dysentery 


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694  Dysentery 

rally,  not  pure;  they  contain  various  amebas  and  numerous 
bacteria. 

To  isolate  and  cultivate  a  single  kind  of  ameba  Musgrave 
and  Clegg  have  recommended  an  ingenious  technic.  A  plate 
is  selected  upon  which  the  desired  amebas  are  so  widely  sep- 
arated from  one  another  that  not  more  than  one  is  in  a 
microscopic  field  of  a  low-power  objective.  The  microscope 
used  should  have  a  double  or  triple  nose-piece.  With  a  low- 
power  (Zeiss  A  A)  objective  a  well  isolated  organism  is  brought 
to  the  center  of  the  field.  The  lens  is  then  swung  out  and  a 
perfectly  clean  higher  power  lens  (Zeiss  DD)  swung  in  and 
racked  down  until  it  touches  the  surface  of  the  agar-agar,  when 
it  is  quickly  elevated  again.  In  three  out  of  five  cases  the 
ameba  adheres  to  the  objective  and  is  so  picked  up.  Whether 
it  has  done  so  or  not  can  be  determined  by  swinging  in  the 
low-power  lens  again  and  looking  for  the  organism.  If  it 
has  disappeared,  it  is  attached  to  the  objective.  It  is  now 
planted  upon  a  fresh  plate  by  depressing  the  high-power  lens 
until  it  touches  the  surf  ace  of  the  culture-medium,  when,  upon 
elevating  it  again,  it  usually  leaves  the  ameba  behind.  Ob- 
servation with  the  low  power  will  enable  one  to  determine 
whether  it  be  successfully  planted  or  not. 

Naturally  the  organisms  cannot  be  thus  transplanted 
without  some  bacteria  falling  upon  the  plate,  but  this  is  not 
very  material,  for  in  the  first  place  they  do  not  grow  very 
rapidly  upon  the  medium  used  for  culture,  and  in  the 
second,  they  are  essential  to  the  nourishment  of  the  ameba, 
which  is  holophagous,  and  cannot  live  by  the  absorption 
of  nutritious  fluids.  Later  it  was  by  Tsugitani*  shown 
that  killed  cultures  of  bacteria  could  supply  the  necessary 
nourishment.  All  cultures  of  amebas  must  contain  the 
symbiotic  organisms  upon  which  the  amebas  live.  It  can- 
not always  be  foretold  what  symbiotic  organisms  are  needed. 
When  planted  as  above  suggested  a  variety  of  organisms 
grow,  and  as  the  amebas  multiply  and  gradually  extend 
over  the  plate,  their  preference  for  one  or  other  of  the 
associated  bacteria  may  be  determined  in  part  by  placing  a 
drop  of  the  ameba  culture  upon  a  plate  of  sterile  media,  and 
then  with  the  platinum  wire,  dipped  in  a  culture  of  the  bac- 
teria, drawing  concentric  circles  about  the  drop  further  and 
further  apart.  As  the  amebas  move  about  over  the  plate, 
passing  through  the  growing  circles  of  bacteria,  they  soon 
*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Abt.  5,  xxiv,  666. 


Amebic  Dysentery  695 

loose  the  miscellaneous  bacteria  and  come  to  contain  the  one 
variety  planted  with  them,  or  if  several  have  been  used  in 
drawing  different  circles,  that  one  which  they  prefer  to  feed 
upon.  By  transplanting  the  amebas  from  plate  to  plate  with 
suitable  symbiotic  bacteria  for  them  to  feed  upon,  the  cul- 
tures may  be  kept  growing  almost  indefinitely. 

Anna  Williams*  has  been  able  to  grow  ameba  in  pure 
culture  without  symbiotic  bacteria,  either  dead  or  alive,  by 
smearing  the  surface  of  a  freshly  prepared  agar-agar  plate 
with  a  fragment  of  freshly  removed  rabbit's  or  guinea-pig's 
brain,  kidney,  or  liver,  held  in  a  pair  of  forceps.  The 
ameba  gladly  take  up  and  live  upon  the  cells  left  behind 
upon  the  surface  of  the  agar. 

Vital  Resistance. — The  free  amebas  in  the  intestinal  dis- 
charges are  easily  destroyed  by  dilute  germicides  and  by 
drying.  Encysted  amebas  are,  however,  more  difficult  to 
kill.  They  resist  drying  well  and  also  resist  the  penetra- 
tion of  germicides.  Direct  sunlight  inhibits  the  activities 
of  the  organisms,  but  does  not  kill  them.  Losch  was  the 
first  to  observe  that  quinin  was  destructive  to  intestinal 
amebas,  and  his  observations  have  been  reviewed  by  many 
others.  Musgrave  and  Clegg  found  that  active  cultures  of 
one  ameba  were  killed  in  ten  minutes  by  a  i  :  2500  solution 
of  quinin  hydrochlorate.  The  exposed  organisms  quickly 
encysted  themselves,  and  in  from  five  to  eight  minutes  many 
of  them  had  broken  up  and  disappeared.  After  ten  minutes 
all  were  dead.  Cultures  of  another  ameba  similarly  treated 
gave  a  scanty  growth  after  ten  minutes. 

Exposure  to  i  :  1000  solution  of  formalin  did  not  kill  en- 
cysted amebas  in  twenty-four  hours.  Acetozone  did  not  kill 
amebas  in  i  :  1000  dilutions.  If,  however,  the  acetozone  was 
made  i  per  cent,  acid  to  phenolphthalein  the  amebas  were  all 
killed  by  i  :  5000  solutions  in  ten  minutes. 

Metabolic  Products. — It  seems  as  though  Entamoeba  his- 
tolytica  must  produce  some  metabolic  product  that  exerts  an 
enzymic  action  upon  the  human  tissues  and  thus  account  for 
the  destructive  nature  of  the  lesions.  This  has  not,  however, 
been  demonstrated  as  yet. 

Pathogenesis. — Schaudinn  was  the  first  to  prove  the  patho- 
genic action  of  the  organism.  He  inspissated  the  evacuations 
of  a  case  suffering  from  dysentery,  so  that  it  contained  con- 
siderable numbers  of  encysted  amebas.  When  this  was  fed 
!  "Journal  of  Medical  Research,"  xxv,  No.  2,  Dec..  1911,  p.  263. 


696 


Dysentery 


to  kittens  they  died  in  two  weeks  with  the  typical  lesions  of 
dysentery.  Musgrave  and  Clegg  had  less  satisfactory  re- 
sults with  cats,  dogs,  and  other  laboratory  animals,  but  were 
quite  satisfied  of  the  results  secured  with  monkeys,  which 
took  the  disease  and  sometimes  died.  The  lesions  resembled, 


but  were  less  severe  than,  those  in  man.  Musgrave  and  Clegg 
would  not  admit  that  there  were  non-pathogenic  intestinal 
amebas,  but  this  was  not  in  accord  with  the  work  of  any  other 
investigators,  and  was  strongly  opposed  by  Craig,*  who  found 
both  varieties,  and  though  he  was  never  able  to  infect  ani- 
*  "Journal  of  Infectious  Diseases,"  v,  1908,  p.  324. 


Amebic  Dysentery  697 

mals  with  Emtamoeba  coli,  was  successful  with  the  pathogenic 
varieties,  and  succeeded  in  infecting  50  per  cent,  of  the  kittens 
he  experimented  upon  by  injecting  the  amebas  into  the 
rectum. 

Lesions. — The  gross  morbid  appearances  of  the  intestinal 
lesions  in  both  forms  of  dysentery  are  sufficiently  distinct  in 
typical  cases  to  enable  an  experienced  pathologist  to  differ- 
entiate them,  yet  not  sufficiently  distinct  to  make  them  easy 
of  description.  The  one  great  characteristic  feature  of  the 
amebic  dysentery  is  abscess  of  the  liver  which  occurs  in 
nearly  25  per  cent,  of  the  cases,  but  which  never  occurs  in 
bacillary  dysentery. 

The  distinct  and  somewhat  rigid  ectoplasm  of  the  Enta- 
moeba  histolytica  is  supposed  to  make  it  easy  for  the  organism, 
which  it  will  be  remembered  are  actively  motile,  to  penetrate 
between  the  epithelial  cells  of  the  intestinal  mucosa  to  the 
lymph-spaces  of  the  submucosa  below.  Here  the  amebas 
multiply  in  large  numbers,  and  by  the  enzymic  action  of  their 
metabolic  products  produce  necrosis  of  the  suprajacent  tis- 
sues with  resulting  exfoliation  and  the  production  of  round, 
oval,  or  ragged  ulcerations  with  markedly  infiltrated  and  un- 
dermined edges.  As  the  amebas  continue  to  increase  and 
fill  up  the  lymphatics,  and  as  bacteria  add  their  effects  to 
those  occasioned  by  the  amebas,  the  ulcers  increase  in  extent 
and  depth  until  the  mucosa  and  submucosa  may  be  almost 
entirely  destroyed,  leaving  the  entire  large  intestine  denuded, 
except  for  occasional  islands  of  much  congested,  inflamed,  and 
partly  necrotic  mucous  membrane.  The  diseased  wall  is  the 
seat  of  much  congestion  and  is  much  thickened.  The 
amebas  not  only  occur  in  great  numbers  in  the  interstices 
of  the  tissues  about  the  base  of  the  ulcers  and  in  the  lym- 
phatics, but  also  enter  the  capillaries,  through  which  they  are 
carried  to  the  larger  vessels,  and  eventually  to  the  liver,  where 
their  activities  continue  and  give  rise  to  the  amebic  abscess. 
The  first  expression  of  their  injury  to  the  liver  parenchyma 
is  shown  by  focal  necroses.  In  each  of  these  the  organisms 
multiply  and  the  lesion  extends  until  neighboring  necroses  are 
brought  into  union,  and  eventuate  in  great  collections  of 
colliquated  necrotic  material  which  may  be  so  extensive  as 
to  involve  the  entire  thickness  of  the  organ.  There  is  usually 
one  large  abscess,  but  there  may  be  several  small  ones,  or 
the  liver  may  be  riddled  with  minute  lesions.  The  contents 
of  the  abscess  is  pinkish  necrotic  material  in  which  amebas  are 
few.  The  walls  are  of  semi- necrotic  material,  in  which  great 


698  Dysentery 

numbers  of  amebas  abound.  The  liver  sometimes  becomes 
adherent  to  the  diaphragm,  may  perforate  it,  and  after  ad- 
hesion of  the  lung  to  the  diaphragm  may  evacuate  through 
the  lung,  the  pinkish  abscess  contents  with  amebas  being 
expectorated. 


m  ***.:• 

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^°^a. 


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% 


W 


v 
X 


Fig.  233. — Entamoeba  histolytica.  Section  of  the  human  intestinal 
wall  showing  the  amebas  at  the  base  of  a  dysenteric  ulcer:  A,  A,  A, 
Amebas,  some  of  which  are  in  blood-vessels,  Gf  (Harris). 

Sections  of  the  intestinal  wall  and  of  the  liver  near  the 
border  of  the  abscess  show  the  amebas  well  when  stained 
with  iron-hematoxylon,  or  perhaps  still  better  by  Mallory's 
differential  method.* 

1.  Harden  the  tissue  in  alcohol. 

2.  Stain  sections  in  a  saturated  aqueous  solution  of  thionin  three  to 
five  minutes. 

3.  Differentiate  in  a  2  per  cent,  aqueous  solution  of  oxalic  acid  for 
one-half  to  one  minute. 

4.  Wash  in  water. 

5.  Dehydrate  in  absolute  alcohol. 

6.  Clear  in  alcohol. 

7.  Xylol-balsam. 

The  nuclei  of  the  amebas  and  the  granules  of  the  mast-cells  are 
stained  brownish  red ;  the  nuclei  of  the  mast-cells  and  of  all  other  cells 
are  stained  blue. 

*"  Pathological  Technic,"  1911,  p.  434. 


Bacillary  Dysentery  699 

IL   BACILLARY  DYSENTERY. 

BACILLUS  DYSENTERIC  (SHIGA), 

General  Characteristics. — A  non-motile,  non-flagellated,  non- 
sporogenous,  non-liquefying,  aerobic  and  optionally  anaerobic,  non- 
chromogenic,  non-aerogenic,  pathogenic  bacillus  of  the  intestine, 
staining  by  ordinary  methods,  but  not  by  Gram's  method.  It  does 
not  produce  mdol.  It  first  acidifies,  then  alkalinizes  milk,  but  does  not 
coagulate  it. 

After  considerable  investigation  of  the  epidemic  dysentery 
prevalent  in  Japan,  Shiga*  has  come  to  the  conclusion  that 
a  bacillus  which  he  calls  Bacillus  dysenteriae  is  its  specific 
cause. 

It  is  not  improbable  that  the  bacillus  of  Shiga  is  identical 
with  Bacterium  coli,  variety  dysenteries,  of  Celli,  Fiocca,  and 
Scala,t  a  view  that  has  been  further  confirmed  by  Flexner.J 
It  may  also  be  identical  with  an  organism  described  in  1888 
by  Chantemasse  and  Widal.§ 

In  1899  Flexner,||  while  visiting  the  Philippine  Islands,  iso- 
lated a  bacillus  from  the  epidemic  dysentery  prevailing  there, 
which  he  regarded  as  identical  with  Shiga's  organism.  In 
1890  Strong  and  Musgrave**  isolated  what  appeared  to  be 
the  same  organism,  also  from  cases  of  dysentery  in  the 
Philippines.  Almost  at  the  same  time  Kruseft  was  inves- 
tigating an  epidemic  of  dysentery  in  Germany,  and  suc- 
ceeded in  isolating  a  bacillus  that  also  bore  fair  correspond- 
ence to  that  of  Shiga.  In  1901  SpronckJt  found  a  bacillus  in 
cases  of  dysentery  occurring  in  Utrecht,  Holland,  that  cor- 
responded with  a  slightly  different  organism  first  found  and 
described  by  Kruse§§  as  a  "  pseudodysentery  bacillus." 

In  1902  Park  and  Dunham ||||  investigated  a  small  epi- 
demic of  dysentery  in  Maine,  and  there  found  a  bacillus 
similar  to  those  already  described.  In  1903  Hiss  and  Rus- 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1898,  xxiv,  Nos.  22-24. 

f'Hygien.  Institut.  Rom.  Univ.,"  1895,  and  "Centralbl.  f.  Bakt. 
u.  Parasitenk.,"  1899. 

|  "Univ.  of  Penna.  Med.  Bulletin,"  Aug.,  1901. 

§  "See  Deutsche  med.  Wochenschrift,"  1903,  No.  12. 

||  "Bulletin  of  the  Johns  Hopkins  Hospital,"  1900,  ix. 
**  "Report  Surg.  Gen.  U.  S.  Army,"  Washington,  1900. 
ff  "  Deutsche  med.  Wochenschrift,"  1900,  xxvi. 
tt  "  Ref.  Baumgarten's  Jahresberichte,"  1901. 
§§  "  Deutsche  med.  Wochenschrift,"  1901,  Nos.  23  and  24. 
||||  "New  York  Bull,  of  Med.  Sciences,"  1902. 


700  Dysentery 

sell  described  a  bacillus  "  Y  "  from  a  case  of  fatal  diarrhea  in 
a  child. 

Bacillus  dysenteriae  was  also  found  by  Vedder  and  Duval* 
in  the  epidemic  and  sporadic  dysentery  of  the  United  States. 
Duval  and  Bassettf  and  Martha  WollsteinJ  found  Bacillus 
dysenteriae  in  cases  of  the  summer  diarrheas  of  infants,  especi- 
ally when  such  diarrheas  were  epidemic. 

About  this  time  Lentz§  published  an  interesting  and  im- 
portant paper  in  which  such  dysentery  and  pseudodysentery 
bacilli  as  he  could  secure  were  found  to  present  differences 
in  their  behavior  toward  sugars.  Other  observers  were  care- 
fully comparing  the  behavior  of  the  various  bacilli  by  means 
of  the  agglutination  by  artificially  prepared  immune  serum. 
The  outcome  of  these  investigations  is  the  discovery  that 
Bacillus  dysenteriae  is  a  species  in  which  there  are  a  number 
of  different  varieties  well  characterized,  but  by  differences 
too  slight  to  permit  them  to  be  regarded  as  separate  species. 
This  thought — that  we  are  dealing  with  a  group  of  varieties 
and  not  a  single  well-defined  organism — is  essential  to  an 
intelligent  understanding  of  the  bacteriology  of  dysentery. 

Before  taking  up  the  variations,  the  characters  common  to 
all  and  constituting  those  of  the  type  species  must  be 
described. 

Morphology. — The  organism  is  a  short  rod  with  rounded 
ends,  generally  similar  to  the  typhoid  bacilli.  It  usually 
occurs  singly,  but  may  occur  in  pairs.  It  is  frequently  sub- 
ject to  involutional  changes.  It  is  doubtfully  motile  and  is 
probably  without  flagella. 

Staining. — When  stained  with  methylene-blue  the  ends 
color  more  deeply  than  the  middle;  and  organisms  from 
old  cultures  show  numerous  involution  forms  and  irregu- 
larities. It  stains  with  ordinary  solutions,  but  not  by 
Gram's  method.  It  has  no  spores. 

Cultivation. — The  organism  grows  well  in  slightly  al- 
kaline media  under  aerobic  conditions. 

Colonies. — The  colonies  upon  gelatin  plates  are  small  and 
dewdrop-like  in  appearance.  Upon  microscopic  examination 
they  are  seen  to  be  regular  and  of  spheric  form.  By  trans- 

*  "Journal  of  Experimental  Medicine,"  vol.  vi,  No.  2,  1902;  "Amer- 
ican Medicine,"  1902. 

t  "American  Medicine,"  Sept.  13,  1902,  vol.  iv,  No.  n,  p.  417. 
J  "Jour.  Med.  Research,"  x,  p.  u,  1904. 
§  "Zeitschrift  f.  Hygiene,"  etc.,  1902,  xu. 


Bacillary  Dysentery  701 

mitted  light  they  appear  granular  and  of  a  yellowish  color. 
They  do  not  spread  out  in  a  thin  pellicle  like  those  of  the 
colon  bacillus,  and  there  are  no  essential  differences  between 
superficial  and  deep  colonies. 

Gelatin  Punctures. — The  growth  in  the  puncture  culture 
consists  of  crowded,  rounded  colonies  along  the  puncture. 
A  grayish- white  growth  forms  upon  the  surface.  There  is 
no  liquefaction  of  the  gelatin. 

Agar-agar. — Upon  the  surface  of  agar-agar,  cultures 
kept  in  the  incubating  oven  show  large  solitary  colonies  at 
the  end  of  twenty-four  hours.  They  are  bluish- white  in 
color  and  rounded  in  form.  The  surface  appears  moist. 
In  the  course  of  forty-eight  hours  a  transparent  border  is 
observed  about  each  colony,  and  the  bacilli  of  which  it  is 
composed  cease  to  stain  evenly,  presenting  involution  forms. 

Glycerin  agar-agar  seems  less  well  adapted  to  their  growth 
than  plain  agar-agar.  Blood-serum  is  not  a  suitable  medium. 

Litmus  Milk. — Milk  is  not  coagulated.  As  the  growth 
progresses  there  is  slight  primary  acidity,  which  later  gives 
place  to  an  increasing  alkalinity. 

Potato. — Upon  boiled  potato  the  young  growth  resembles 
that  of  the  typhoid  bacillus,  but  after  twenty-four  hours  it 
becomes  yellowish  brown,  and  at  the  end  of  a  week  forms 
a  thick,  brownish-pink  pellicle. 

Bouillon. — In  bouillon  the  bacillus  grows  well,  clouding 
the  liquid.  No  pellicle  forms  on  the  surface. 

Metabolic  Products. — The  organism  does  not  form  in- 
dol,  does  not  ferment  dextrose,  lactose,  saccharose,  or  other 
carbohydrates.  Acids  are  produced  in  moderate  quantities 
after  twenty-four  hours.  Milk  is  not  coagulated.  Gelatin 
is  not  liquefied. 

Toxins,  chiefly  endotoxins,  are  produced.  They  may  best 
be  prepared  by  making  massive  agar-agar  cultures  in  Kitasato 
flasks  or  flat-sided  bottles,  and  after  growth  is  complete 
washing  off  the  bacillary  mass  with  a  very  small  quantity  of 
sterile  salt  solution,  and  after  killing  the  bacilli  by  exposure 
to  60°  C.  for  fifteen  to  thirty  minutes,  permitting  the  rich 
suspension  to  autolyze  for  three  days.  The  toxins  may  be 
precipitated  from  the  sodium  chlorid  solution  by  ammonium 
sulphate. 

Vital  Resistance. — The  thermal  death-point  is  68°  C. 
maintained  for  twenty  minutes.  It  grows  slowly  at  ordi- 
nary temperatures,  rapidly  at  the  temperature  of  the  body. 


702  Dysentery 

The  Different  Varieties  of  the  Dysentery  Bacillus.— 

Three  varieties  of  the  dysentery  bacillus  may  now  be  de- 
scribed : 

1.  The  Shiga-Kruse  variety. 

2.  The  Flexner  variety. 

3.  The  Hiss-Russell  variety. 

The  differences  by  which  they  are  separated  are  to  be 
found  in  the  agglutinability  by  artificially  prepared  immune 
serum,  each  of  which  exerts  a  far  more  pronounced  effect 
upon  its  own  variety  than  upon  the  others,  and  in  the  be- 
havior toward  sugars  with  reference  to  acid  formation  and 
gas  production.  It  seems  not  improbable  that  the  future 
will  have  much  to  say  about  the  dysentery  bacillus,  and 
that  the  validity  of  much  that  is  accepted  at  present  may 
have  to  be  amended.  This  seems  to  be  particularly  true 
with  regard  to  the  matter  of  fermentation,  the  details  of 
which  are  displayed  in  the  following  table  taken  from  Muir 
and  Ritchie's  "Manual  of  Bacteriology." 

Pathogenesis. — Shiga  and  Flexner  found  that  infection 
of  young  cats  and  dogs  could  be  effected  by  bacilli  introduced 
into  the  stomach,  and  that  lesions  suggestive  of  human  dys- 
entery were  found  in  the  intestines.  Kazarinow  *  found  that 
when  guinea-pigs  and  young  rabbits  were  narcotized  with 
opium,  the  gastric  contents  alkalinized  with  10  c.c.  of  a  10 
per  cent.  NaOH  solution,  and  a  quantity  of  Shiga  bacilli 
introduced  into  the  stomach  with  an  esophageal  bougie,  it 
was  possible  to  bring  about  diarrhea  and  death  with  lesions 
similar  to  those  described  by  Vaillard  and  Dopter. 

In  these  experiments  it  was  found  that  rapid  passage 
through  animals  greatly  increased  the  virulence  of  the 
bacilli,  and  it  was  also  observed  that  though  0.0005  c.c.  of 
a  virulent  culture  introduced  into  the  peritoneal  cavity 
would  cause  fatal  infection,  to  produce  infection  by  the 
mouth  as  above  stated  required  the  entire  mass  of  organisms 
grown  in  five  whole  culture- tubes. 

The  virulent  organisms  are  infectious  for  guinea-pigs  and 
other  laboratory  animals,  and  cause  fatal  generalized  infec- 
tion without  intestinal  lesions. 

Lesions. — The  lesions  found  in  human  dysentery  are 
usually  fairly  destructive.  They  consist  of  a  severe  catarrhal 
and  pseudomembranous  colitis,  which  later  passes  into  a 

*  " Archiv.  f.  Hyg.,"  Bd.  L,  Heft  i,  p.  66;  see  also  "Bull,  de  1'Inst. 
Past.,"  15  Aout,  1904,  p.  634. 


Bacillary  Dysentery 


703 


* 

o 
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Reaction. 

704  Dysentery 

stage  of  marked  ulceration.  There  is  great  thickening  of  the 
submucosa  and  the  whole  of  the  intestinal  lining  is  corru- 
gated. For  the  most  part  the  ulcerations  are  more  superfi- 
cial than  those  of  the  amebic  dysentery,  and  the  edges  of  the 
ulcerations  show  less  tumefaction  and  less  undermining. 
Abscess  of  the  liver  does  not  occur  in  bacillary  dysentery. 

Diagnosis. — The  blood-serum  of  those  suffering  from 
epidemic  dysentery  or  from  those  recently  recovered  from  it 
causes  a  well-marked  agglutinative  reaction.  This  agglutina- 
tion was  first  carefully  studied  by  Flexner,  and  is  peculiar, 
in  that  the  serums  prepared  from  the  different  varieties  of 
the  bacillus,  while  they  exert  some  action  upon  all  varieties 
of  the  organism,  exert  a  much  more  powerful  influence  upon 
the  particular  variety  used  in  their  preparation.  The  same 
is  true  of  the  patient's  serum,  hence,  in  making  use  of  the 
agglutination  reaction  for  the  diagnosis  of  the  disease,  the 
blood  of  the  patient  should  be  tested  by  contact  with  all  of 
the  different  cultures. 

Serum  Therapy. — By  the  progressive  immunization  of 
horses  to  an  immunizing  fluid,  the  basis  of  which  is  a  twenty- 
four-hour-old  agar-agar  culture  dried  in  vacua,  Shiga  has 
prepared  an  antitoxic  serum  with  which,  in  1898,  in  the 
Laboratory  Hospital,  65  cases  were  treated,  with  a  death-rate 
of  9  per  cent.;  in  1899,  in  the  Laboratory  Hospital,  91  cases, 
with  a  death-rate  of  8  per  cent.;  in  1899,  in  the  Hirowo 
Hospital,  no  cases,  with  a  death-rate  of  12  per  cent.  These 
results  are  very  significant,  as  the  death-rate  in  2736  cases 
simultaneously  treated  without  the  serum  averaged  34.7 
per  cent.,  and  in  consideration  of  the  frequency  and  high 
death-rate  of  the  disease,  Japan  alone,  between  the  years 
1878  and  1899,  furnishing  a  total  of  1,136,096  cases,  with 
275,308  deaths  (a  total  mortality  for  the  entire  period  of 
24.23  per  cent.).* 


III.   BALANTIDIUM  DIARRHEA. 
BALANTIDIUM  Cou  (MALMSTEN). 

In  certain  rare  cases  a  severe  form  of  diarrhea,  or  a  mild 

form  of  dysentery  appears  to  depend  neither  upon  Entamoeba 

histolytica  nor  Bacillus  dysenteriae,  but  upon  an  infusorian 

parasite  known  as  Balantidium  coli.     This  organism  was 

*  "Public  Health  Reports,"  Jan.  5,  1900,  vol.  xv,  No.  i. 


Balantidium  Diarrhea  705 

first  observed  by  Malmsten*  in  1857  in  the  intestines  of  a 
man  who  had  suffered  from  cholera  two  years  before  and  had 
ever  since  suffered  from  diarrhea.  Upon  investigation  an 
ulceration  was  found  in  the  rectum  just  above  the  internal 
sphincter.  In  the  bloody  pus  from  this  ulcer  numerous 
balantidia  were  seen  swimming  about.  Although  the  ulcer 
healed,  the  diarrhea  did  not  cease.  Since  this  original  ob- 
servation and  up  to  1908  Braunf  had  been  able  to  collect 
142  cases  of  human  infection.  In  all  of  these  cases  the  pres- 
ence of  the  balantidium  was  accompanied  by  obstinate  diar- 
rhea with  bloody  discharges  (dysentery)  in  some,  and  many 
of  the  cases  ended  in  death. 

Morphology. — The  Balantidium  coli  is  an  infusorian 
micro-organism  of  ovoid  or  ellipsoidal  form,  measuring  from 
30  to  200  fi  in  length  and  from  20  to  70  [i  in  breadth.  The 
body  is  surrounded  by  a  distinct  ectosarc  completely  covered 
by  short  fine  cilia.  The  anterior  end,  which  is  usually  a  little 
sharper  than  the  posterior,  presents  a  deep  indentation,  the 
peristome,  which  continues,  in  an  infundibuliform  manner, 
deeply  into  the  endosarc.  The  peristome  is  surrounded  by  a 
circle  of  longer  cilia — adoral  cilia — than  those  elsewhere 
upon  the  body.  At  the  opposite  pole  there  is  a  small  open- 
ing in  the  ectosarc,  the  anus.  The  mouth  is  the  simple  ter- 
mination of  the  infundibuliform  extension  of  the  peristome 
and  opens  directly  into  the  endosarc,  so  that  the  small  bodies 
upon  which  the  organism  feeds,  and  which  are  continually 
being  caught  in  the  vortex  caused  by  the  rapidly  vibrating 
adoral  cilia  are  driven  down  the  short  tubulature  directly 
into  the  endosarc. 

The  endosarc  is  granular  and  contains  fat  and  mucin 
granules,  starch  grains,  bacteria,  and  occasionally  red  and 
white  blood-corpuscles. 

There  are  usually  two  contractile  vacuoles,  sometimes  more, 
and  as  the  quiet  organism  is  watched  these  large  clear  spaces 
can  be  seen  alternately  to  contract  and  expand. 

There  are  two  nuclei.  The  larger,  or  macronucleus,  is  bean- 
shaped,  kidney-shaped,  or,  more  rarely,  oval.  The  smaller, 
the  micronucleus,  is  spherical.  There  is  no  digestive  tube; 
the  nutritious  particles  are  directly  in  the  endosarc,  in  which 
they  are  digested,  any  residuum  being  extruded  from  the 
anus. 

!  "Archiv.  f.  pathologische  Anatomic,"  etc.,  xn,  1857,  p.  302. 
t  "Tierische  Parasiten  des  Menschen,"  Wiirzburg,  1908. 
45 


706 


Balantidium  Coli 


Motility. — The  organism  is  actively  motile,  swimming 
rapidly  at  a  steady  pace  or  darting  here  and  there. 

Staining. — The  organism  can  be  most  easily  and  satisfac- 
torily studied  while  alive.  To  stain  it  a  drop  of  the  fluid 
containing  the  balantidia  is  spread  upon  a  slide  and  per- 
mitted to  dry.  Just  before  the  moisture  disappears  from  the 
film,  methyl  alcohol  may  be  poured  upon  it  to  kill  and  fix 
the  organisms.  The  staining  may  then  be  performed  with 
Giemsa's  polychrome  methylene-blue  or  iron-hematoxylon. 
The  cilia  usually  do  not  show. 


Fig.  234. — Reproduction  of  Balantidium  coli:  1-5,  Asexual  reproduc- 
tion by  division;  6,  encysted  form  of  single  individuals;  7,  conjugation 
of  two  individuals;  8,  reproductive  cyst;  9,  cyst  with  peculiar  contents 
whose  further  development,  has  not  been  followed  (Brumpt). 

Reproduction. — This  commonly  takes  place  by  karyoki- 
nesis,  followed  by  transverse  division,  and  in  cases  of  ex- 
perimental infection  so  rapidly  that  the  organisms  have  not 
time  to  grow  to  the  full  size  before  dividing  again.  The  result 
is  that  many  appear  that  are  no  more  than  30  (*  in  length. 
In  addition  to  multiplication  by  division,  there  is  a  sexual 
cycle  of  development  with  conjugation.  This  was  first  pointed 


Balantidium  Diarrhea  707 

out  by  Gourvitsch,*  studied  by  Leger  and  Duboscq,f  and 
further  confirmed  by  BrumptJ.  In  the  process  of  conjuga- 
tion two  individuals  come  together,  become  attached  length- 
wise, and  fuse  into  a  single  large  organism  that  forms  a  cyst 
several  times  as  large  as  a  balantidium,  and  with  contents  no 
longer  recognizable  as  such.  The  contents  of  this  cyst 
eventually  divide  into  a  number  of  spheres,  but  how  these 
subsequently  develop  appears  not  to  have  been  deter- 
mined. 

Habitat. — The  balantidium  is  unknown  except  as  a  para- 
site of  the  colon.  It  is  very  common  in  hogs  and  has  been 
found  in  the  orang-outang,  in  certain  lower  monkeys 
(Macacus  cynomolgus),  and  in  man. 

Cultivation. — The  organism  quickly  dies  when  trans- 
planted to  artificial  media  and  has  not  yet  been  cultivated 
artificially. 

Pathogenesis. — The  presence  of  the  organisms,  in  whatever 
kind  of  animal,  gives  rise  to  colitis,  which  is  at  first  catarrhal, 
but  soon  becomes  more  or  less  ulcer ative.  Some  doubt  has 
been  expressed  as  to  the  exact  role  of  the  balantidia  in 
the  causation  of  the  inflammation,  some  believing  them  to  be 
rather  accidental  factors  than  the  true  etiologic  excitants. 
As  the  organisms  descend  into  the  ulcerated  tissues  and  from 
the  denuded  surfaces  invade  the  lymphatics,  there  seems  to 
be  little  doubt  of  their  pathogenic  activity. 

Animal  Inoculation. — Experiments  made  by  Casagrandi 
and  Barbagallo,§  Klimenko,  ||  and  others  upon  kittens  and 
pups  have  failed  to  produce  the  disease  even  when  the  colon 
was  already  inflamed.  Brumpt,**  on  the  contrary,  succeeded 
in  reproducing  it  in  monkeys  and  pigs  by  introducing  the 
encysted  organisms  into  the  already  inflamed  intestine  via  the 
anus. 

Lesions. — In  the  majority  of  fatal  cases  postmortem  ex- 
amination of  the  colon  shows  it  to  be  in  a  state  of  catarrhal 
inflammation  with  numerous  superficial  ulcerations  with  con- 
siderable surrounding  infiltration  of  the  mucosa.  Twenty- 

*"Russ.  Archiv.  f.  Path.  klin.  Med.  u.  Bact.  St.  Petersb.,"  1896, 
quoted  by  Braun. 

t  "Archiv.  de  Zool.  Exper.,"  1904,  n,  No.  4. 
J  "Compt.-rendu  de  la  Soc.  de  Biol.,"  July  10,  1909. 
§  "Bal.  coli,"  etc.,  Catania,  1896,  quoted  by  Braun. 
||  "Beitrage  zur.  Path.  Anat.  u.  allg.  Path.,"  1903,  xxxn,  281. 
**  "Precis  de  Parasitology,"  1910,  152. 


Balantidium  Coli 


four  hours  from  the  time  of  the  death  of  the  patient  the 
balantidia  are  all  dead.  Strong  and  Musgrave,*  Solowiew,  f 
Klimenko,  t  and  others  have  shown  that  in  microscopic  sec- 
tions of  the  inflamed  tissues  the  micro-organisms  could  be 
found  deep  down  in  the  blood-vessels  and  lymphatic  spaces 
about  the  ulcerated  areas,  sometimes  penetrating  as  deeply 
as  the  serous  coat  of  the  bowel.  Metastatic  abscess  of  the 
liver  may  be  caused  by  balantidia,  and  has  been  reported 


Fig-  235. — Balantidium  coli  deeply  situated  in  the  interglandular  tissue 
of  the  intestinal  mucosa  (Brumpt). 

by  Manson,§  and  a  case  of  abscess  of  the  lung  caused  by 
the  organism  by  Winogradow  and  Stokvis.H 

Transmission. — The  transmission  of  the  disease  can  only 
come  about  through  the  encysted  form  of  the  parasites. 
Great  numbers  are  passed  in  the  fecesof  the  infected  animals, 

*  "Bulletin  of  the  Johns  Hopkins  Hospital,"  1901,  xn,  31. 
t  "Centralbl.  f.  Bakt.,"  etc.,  i  Abl.,  1901,  xxix,  821,  849. 
t  Loc.  cit. 

§  "Tropical  Diseases,"  1900,  p.  394. 

||  "Niederl.  Tijdschr.  v.  Geneeskde.,"  1884,  xx,  No.  2,  quoted  by 
Braun. 


Balantidium  Diarrhea  709 

but  except  the  encysted  forms  all  die  very  quickly  as  the 
fecal  matter  dies.  Unfortunately  the  further  life-history  of 
the  encysted  forms  is  unknown. 

FLAGELLATES  IN  THE  HUMAN  INTESTINES. 

In  certain  cases  of  diarrhea,  flagellates — Trichimonas  in- 
testinalis,  Cercomonas  intestinalis,  and  Lamblia  (Megasto- 
mum)  intestinalis  have  been  discovered.  As,  however,  they 
seem  to  be  frequent  denizens  of  normal  intestines,  it  is 
doubtful  whether  their  presence  is  more  than  incidental. 


CHAPTER  XXVII. 
TUBERCULOSIS. 

BACILLUS  TUBERCULOSIS  (KOCH).* 

General  Characteristics. — A  non-motile,  non-flagellate,  non-spor- 
ogenous,  non-liquefying,  non-chromogenic,  non-aerogenic,  distinctly 
aerobic,  acid-resisting,  purely  parasitic,  highly  pathogenic  organism, 
staining  by  special  methods  and  by  Gram's  method.  Commonly  oc- 
curring in  the  form  of  slender,  slightly  curved  rods  with  rounded  ends, 
not  infrequently  showing  branches,  hence  probably  not  a  bacillus,  but 
an  organism  belonging  to  the  higher  bacteria.  It  does  not  produce 
indol  or  acidulate  or  coagulate  milk. 

Tuberculosis  is  one  of  the  most  dreadful  and,  unfor- 
tunately, one  of  the  most  common  diseases.  It  is  no  re- 
specter of  persons,  but  affects  alike  the  young  and  old,  the 
rich  and  poor,  the  male  and  female,  the  enlightened  and 
savage,  the  human  being  and  the  lower  animals.  It  is  the 
most  common  cause  of  death  among  human  beings,  and  is 
common  among  animals,  occurring  with  great  frequency 
among  cattle,  less  frequently  among  goats  and  hogs,  and 
sometimes,  though  rarely,  among  sheep,  horses,  dogs,  and 
cats. 

Wild  animals  under  natural  conditions  seem  to  escape 
the  disease,  but  when  caged  and  kept  in  zoologic  gardens, 
even  the  most  resistant  of  them — lions,  tigers,  etc. — are 
said  at  times  to  succumb  to  it,  while  it  is  the  most  common 
cause  of  death  among  captive  monkeys. 

The  disease  is  not  limited  to  mammals,  but  occurs  in  a 
somewhat  modified  form  in  birds,  and  it  is  said  even  at 
times  to  affect  reptiles,  batrachians,  and  fishes. 

The  disease  has  been  recognized  for  centuries ;  and  though, 
before  the  advent  of  the  microscope,  it  was  not  always  clearly 
differentiated  from  cancer,  it  has  not  only  left  unmistakable 
signs  of  its  existence  in  the  early  literature  of  medicine,  but 
has  also  imprinted  itself  upon  the  statute-books  of  some 
countries,  as  the  kingdom  of  Naples,  where  its  ravages  were 
great  and  the  means  taken  for  its  prevention  radical. 

*  "Berliner  klin.  Wochenschrift,"  1882,  15. 
710 


Distribution  711 

Specific  Organism. — Although  the  acute  men  of  the  early 
days  of  pathology  clearly  saw  that  the  time  must  come 
when  the  parasitic  nature  of  tuberculosis  would  be  proved, 
and  Klebs,  Villemin,  and  Cohnheim  were  "  within  an  ace  " 
of  its  discovery,  and  Baumgarten*  probably  saw  it  in  tissues 
cleared  with  lye,  it  remained  for  Robert  Koch  to  demon- 
strate and  isolate  the  Bacillus  tuberculosis,  the  specific 
cause  of  the  disease,  and  to  write  so  accurate  a  description 
of  the  organism,  and  the  lesions  it  produces,  as  to  be  almost 
without  a  parallel  in  medical  literature. 

Distribution. — So  far  as  is  known,  the  tubercle  bacillus  is 
a  purely  parasitic  organism.  It  has  never  been  found 


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Fig.  236. — Tubercle  bacillus  in  sputum  (Frankel  and  Pfeiffer). 

except  in  the  bodies  and  discharges  of  animals  affected  with 
tuberculosis,  and  in  dusts  of  which  these  are  component 
parts.  This  purely  parasitic  nature  interferes  with  the 
isolation  of  the  organism,  which  cannot  be  grown  upon 
the  ordinary  culture-media. 

The  widespread  distribution  of  tuberculosis  at  one  time 
suggested  that  tubercle  bacilli  were  ubiquitous  in  the  at- 
mosphere, that  we  all  inhaled  them,  and  that  it  was  only 
our  vital  resistance  that  prevented  us  all  from  becoming  its 
victims.  Cornet,  f  however,  showed  the  bacilli  to  be  present 

*  "Virchow's  Archives,"  Bd.  LXXXII,  p.  397. 

t  "Zeitschrift  fur  Hygiene,"  v,  1888,  pp.  191-331. 


712 


Tuberculosis 


only  in  dusts  with  which  pulverized  sputum  was  mixed,  and 
to  be  most  common  where  the  greatest  uncleanliness  pre- 
vailed. 

Morphology. — The  tubercle  bacillus  is  a  slender,  rod- 
shaped  organism  with  slightly  rounded  ends  and  a  slight 
curve.  It  measures  from  1.5  to  3.5  [A  in  length  and  from 
0.2  to  0.5  fJ-  in  breadth.  It  commonly  occurs  in  pairs,  which 
may  be  associated  end  to  end,  but  generally  overlap  some- 
what and  are  not  attached  to  each  other.  Organisms  found 
in  old  pus  and  sputum  show  a  peculiar  beaded  appearance 
caused  by  fragmentation  of  the  protoplasm  and  the  presence 


Fig.  237. — Bacillus  of  tuberculosis,  showing  branched  forms  with  invo- 
lution (Migula). 

of  metachromatic  granules.  These  fragmented  forms  have 
been  thought  to  be  bacilli  in  the  stage  of  sporulation,  and 
Koch  originally  held  this  view  himself,  though  later  researches 
have  not  confirmed  it. 

The  tubercle  bacillus  forms  no  endospores.  The  frag- 
ments thought  by  Koch  to  be  spores  are  irregular  in  shape, 
have  ragged  surfaces,  and  are  without  the  high  refraction 
peculiar  to  spores.  Spores  also  resist  heat  strongly,  but  the 
fragmented  bacilli  are  no  more  capable  of  resisting  heat 
than  others. 

The  bacilli  not  infrequently  present  projecting  processes 
or  branches,  this  observation  having  changed  our  views 


Staining  713 

regarding  the  classification  of  the  organism,  which  is  prob- 
ably erroneously  placed  among  the  bacilli,  belonging  more 
properly  to  the  higher  bacteria. 

The  organism  is  not  motile,  and  does  not  possess  flagella. 

Staining. — The  tubercle  bacillus  belongs  to  a  group  of 
organisms  which  because  of  their  peculiar  behavior  toward 
stains  is  known  as  "  saurefest  "  or  acid-proof.  It  is  difficult 
to  stain  after  it  has  lived  long  enough  to  invest  itself  with  a 
waxy  capsule,  requiring  that  the  dye  used  shall  contain  a 
mordant  (Koch).  It  is  also  tenacious  of  color  once  assumed, 
resisting  the  decolorizing  power  of  strong  mineral  acids 
(Ehrlich). 

The  peculiarity  of  staining  the  bacillus  delayed  its  dis- 
covery for  a  considerable  time,  but,  now  that  we  are  familiar 
with  it,  gives  us  a  most  valuable  differential  character,  few 
other  organisms  reacting  in  the  same  way. 

Koch*  first  stained  the  bacillus  with  a  solution  consisting 
of  i  c.c.  of  a  concentrated  solution  of  methylene-blue  mixed 
with  20  c.c.  of  distilled  water,  well  shaken,  and  then,  before 
using,  receiving  an  addition  of  2  c.c.  of  a  10  per  cent,  solu- 
tion of  caustic  potash.  Cover-glasses  were  allowed  to 
remain  in  this  for  twenty-four  hours  and  subsequently 
counterstained  with  vesuvin.  Ehrlich  subsequently  modified 
Koch's  method,  showing  that  pure  anilin  was  a  better 
mordant  than  potassium  hydrate,  and  that  the  use  of  a 
strong  mineral  acid  would  remove  the  color  from  everything 
but  the  tubercle  bacillus.  This  modification  of  Koch's 
method,  given  us  by  Ehrlich,  probably  remains  the  best 
method  of  staining  the  bacillus. 

Nearly  all  of  the  recent  methods  of  staining  are  based 
upon  the  impenetrability  of  the  bacillary  substance  to 
mineral  acids  which  characterizes  the  acid-fast  or  acid-proof 
(saurefest)  micro-organisms.  But  it  is  not  improbable  that 
we  have  been  led  into  error  by  the  assumption,  upon  inade- 
quate grounds,  that  this  is  a  constant  and  uniform  quality 
of  the  tubercle  bacillus  and  similar  micro-organisms.  The 
interesting  observations  of  Muchf  have  shown  that  many  of 
the  paradoxes  of  tuberculosis  can  be  accounted  for  by  the 
fact  that  during  certain  stages,  or  in  certain  conditions,  the 
bacilli  are  not  acid-proof  at  all.  Thus,  examinations  of 
caseous  masses  from  the  lungs  of  cattle  show  complete 

"Mittheilungen  aus  dem  Gesundheitsamte,"  n,  1884. 
t  "Berliner  klin.  Wochenschrift,"  April  6,  1908,  p.  691. 


714  Tuberculosis 

absence  of  tubercle  bacilli  when  examined  by  the  usual 
method,  yet  cause  typical  tuberculosis  when  implanted  into 
guinea-pigs,  with  typical  bacilli,  recoverable  upon  culture- 
media,  in  the  lesions.  This  is  certainly  due  to  the  inability 
of  the  bacilli  in  the  bovine  lesions  mentioned  to  endure  the 
acids,  for  when  the  same  tissues  are  stained  by  Gram's 
method  many  organisms  can  be  found.  This  shows  that 
Gram's  method  is  really  a  more  useful  method  for  demon- 
strating the  bacillus  than  those  in  which  acids  are  employed. 
Naturally,  Gram's  method,  not  being  differential,  is  inappro- 
priate for  sputum,  cavity  contents,  and  tissues  in  which  many 
other  species  of  bacteria  might  be  present.  Much  has  found 
two  forms  of  the  tubercle  bacillus,  one  rod-like,  the  other 
granular,  that  are  not  acid-proof,  and  has  succeeded  in 
changing  one  into  the  other  by  experimental  manipulation. 
He  believes  that  the  acid-proof  condition  has  some  bearing 
upon  virulence,  and  speculates  that  the  more  acid-proof 
the  organisms  are,  the  less  virulent  they  will  be  found. 

In  this  connection  the  work  of  Maher,*  who  claims  to  be 
able,  by  appropriate  methods  of  cultivation,  to  make  many 
of  the  ordinary  saprophytic  bacteria  (Bacillus  coli,  B.  subtilis, 
etc.)  thoroughly  acid-proof,  must  be  mentioned. 

Staining  the  Bacillus  in  Sputum. — As  the  purpose  for 
which  the  staining  is  most  frequently  performed  is  the 
diagnosis  of  the  disease  through  the  demonstration  of  the 
bacilli  in  sputum,  the  method  by  which  this  can  be  accom- 
plished will  be  first  described. 

When  the  sputum  is  mucopurulent  and  nummular,  any 
portion  of  it  may  suffice  for  examination,  but  if  the  patient 
be  in  the  early  stages  of  tuberculosis,  and  the  sputum  is 
chiefly  thin,  seromucus,  and  flocculent,  care  must  be  exercised 
to  see  that  such  portion  of  it  as  is  most  likely  to  contain  the 
micro-organisms  be  examined. 

If  one  desires  to  make  a  very  careful  examination,  it  is 
well  to  have  the  patient  cleanse  the  mouth  thoroughly  upon 
waking  in  the  morning,  and  after  the  first  fit  of  coughing 
expectorate  into  a  clean,  wide-mouthed  bottle,  the  object 
being  to  avoid  the  presence  of  fragments  of  food  in  the 
sputum. 

The  best  result  will  be  secured  if  the  examination  be 
made  on  the  same  day,  for  if  the  bacilli  are  few  they  occur 
most  plentifully  in  small  flakes  of  caseous  matter,  which  are 

*  "  International  Conference  on  Tuberculosis,"  Philadelphia,   1907. 


Staining  715 

easily  found  at  first,  but  which  break  up  and  become  part 
of  a  granular  sediment  that  forms  in  decomposed  sputum. 

The  sputum  should  be  poured  into  a  watch-glass  and 
held  over  a  black  surface.  A  number  of  grayish-yellow, 
irregular,  translucent  fragments  somewhat  smaller  than  the 
head  of  a  pin  can  usually  be  found.  These  consist  prin- 
cipally of  caseous  material  from  the  tuberculous  tissue, 
and  are  the  most  valuable  part  of  the  sputum  for  exam- 
ination. One  of  the  fragments  is  picked  up  with  a  pointed 
match-stick  and  spread  over  the  surface  of  a  perfectly 
clean  cover-glass  or  slide.  If  no  such  fragment  can  be  found, 
the  purulent  part  is  next  best  for  examination. 

The  material  spread  upon  the  glass  should  not  be  too 
small  in  amount.  Of  course,  a  massive,  thick  layer  will  be- 
come opaque  in  staining,  but  should  the  layer  spread  be, 
as  is  often  advised,  "  as  thin  as  possible,"  there  may  be  so 
few  bacilli  upon  the  glass  that  they  are  found  with  diffi- 
culty. 

The  film  is  allowed  to  dry  thoroughly  and  is  then  passed 
three  times  through  the  flame  for  fixation. 

Ehrlich's  Method,  or  the  Kock-Ehrlich  Method. — Cover- 
glasses  thus  prepared  are  floated,  smeared  side  down,  or 
immersed,  smeared  side  up,  in  a  small  dish  of  Ehrlich's 
anilin-water  gentian  violet  solution: 

Anilin 4 

Saturated  alcoholic  solution  of  gentian  violet 1 1 

Water 100 

and  kept  in  an  incubator  or  paraffin  oven  for  about  twenty- 
four  hours  at  about  the  temperature  of  the  body.  Slides 
upon  which  smears  have  been  made  can  be  placed  in  Coplin 
jars  containing  the  stain  and  stood  away  in  the  same  manner. 
When  removed  from  the  stain,  they  are  washed  momentarily 
in  water,  and  then  alternately  in  25  to  33  per  cent,  nitric  acid 
and  60  per  cent,  alcohol,  until  the  blue  color  of  the  gentian 
violet  is  entirely  lost.  A  total  immersion  of  thirty  seconds  is 
enough  in  most  cases.  After  final  thorough  washing  in  60 
per  cent,  alcohol,  the  specimen  is  counterstained  in  a  dilute 
aqueous  solution  of  Bismarck  brown  or  vesuvin,  the  excess  of 
stain  washed  off  in  water,  and  the  specimen  dried  and  mounted 
in  balsam.  The  tubercle  bacilli  are  colored  a  fine  dark  blue, 
while  the  pus-corpuscles,  epithelial  cells,  and  other  bacteria, 
having  been  decolorized  by  the  acid,  will  appear  brown. 


716  Tuberculosis 

This  method,  requiring  twenty-four  hours  for  its  com- 
pletion, has  fallen  into  disuse,  as  it  is  desirable  to  know  in 
the  briefest  possible  time  whether  bacilli  are  present  in  the 
sputum  or  not. 

Ziehl's  Method. — Among  clinicians,  Ziehl's  method  of 
staining  with  carbol-fuchsin  has  met  with  just  favor.  It  is 
as  follows:  After  having  been  spread,  dried,  and  fixed, 
the  cover-glass  is  held  in  the  bite  of  an  appropriate  forceps 
(cover-glass  forceps),  or  the  slide  spread  at  one  end  is  held 
by  the  other  end  as  a  handle,  and  the  stain  (fuchsin,  i ; 
alcohol,  10;  5  per  cent,  phenol  in  water,  100)  dropped  upon  it 
from  a  pipet.  As  soon  as  the  entire  smear  is  covered  with 
stain,  it  is  held  over  the  flame  of  a  spirit  lamp  or  Bunsen 
burner  until  the  stain  begins  to  volatilize  a  little.  When 
vapor  is  observed  the  heating  is  sufficient,  and  the  tem- 
perature can  be  maintained  by  intermittent  heating. 

If  evaporation  take  place,  a  ring  of  encrusted  stain  at  the 
edge  prevents  the  prompt  action  of  the  acid.  To  prevent 
this,  more  stain  should  now  and  then  be  added.  The  stain- 
ing is  complete  in  from  three  to  five  minutes,  after  which 
the  specimen  is  washed  off  with  water,  and  then  with  a  3 
per  cent,  solution  of  hydrochloric  acid  in  70  per  cent,  alco- 
hol, 25  per  cent,  aqueous  sulphuric,  or  33  per  cent,  aqueous 
nitric  acid  solution  dropped  upon  it  for  thirty  seconds,  or 
until  the  red  color  is  extinguished.  The  acid  is  carefully 
washed  off  with  water,  the  specimen  dried  and  mounted 
in  Canada  balsam.  Nothing  will  be  colored  except  the 
tubercle  bacilli,  which  appear  red. 

Gobbet's  Method. — Gabbet  modified  the  method  by 
adding  a  little  methylene-blue  to  the  acid  solution,  which 
he  makes  according  to  this  formula: 

Methylene-blue 2 

Sulphuric  acid '.  .  25 

Water 75 

In  Gabbet 's  method,  after  staining  with  carbol-fuchsinr. 
the  specimen  is  washed  with  water,  acted  upon  by  the 
methylene-blue  solution  for  thirty  seconds,  washed  again 
with  water  until  only  a  very  faint  blue  remains,  dried,  and 
finally  mounted  in  Canada  balsam.  The  tubercle  bacilli 
are  colored  red;  the  pus-corpuscles,  epithelial  cells,  and 
unimportant  bacteria,  blue. 


Staining  717 

Pappenheim,*  having  found  bacilli  stained  red  by  Ziehls' 
method  in  the  sputum  of  a  case  which  subsequent  post- 
mortem examination  showed  to  be  one  of  gangrene  of  the 
lung  without  tuberculosis,  condemns  that  method  as  not 
being  sufficiently  differential,  and  recommends  the  following 
as  superior  to  methods  in  which  the  mineral  acids  are  em- 
ployed : 

1.  Spread  the  film  as  usual. 

2.  Stain  with  carbol-fuchsin,  heating  to  the  point  of  steaming  for  a 

few  minutes. 

3.  Pour  off  the  carbol-fuchsin  and  without  washing — 

4.  Dip  the  spread  from  three  to  five  times  in  the  following  solution, 

allowing  it  to  run  off  slowly  after  each  immersion: 

Corallin i 

Absolute  alcohol 100 

Methylene-blue .  ad  sat. 

Glycerin 20 

5.  Wash  quickly  in  water. 

6.  Dry. 

7.  Mount. 

The   entire   process   takes   about   three   minutes.     The   tubercle 
bacilli  alone  remain  red. 

Where  examination  by  this  means  fails  to  reveal  the 
presence  of  bacilli  because  of  the  small  number  in  which  they 
occur,  recourse  may  be  had  to  the  use  of  caustic  potash  or, 
what  is  better,  antiformin  (q.  v.)  for  digesting  the  sputum. 
A  considerable  quantity  of  sputum  is  collected,  receives  the 
addition  of  an  equal  volume  of  the  antiformin,  is  permitted 
to  stand  until  the  formed  elements  and  pus-corpuscles  have 
been  dissolved,  is  then  shaken  and  poured  into  centrifuge 
tubes  and  whirled  for  fifteen  to  thirty  minutes.  The  sedi- 
ment at  the  bottom  of  the  tubes  will  then  reveal  the  bacilli 
which,  having  been  freed  from  the  viscid  materials  in  the 
sputum,  have  been  thrown  down  by  the  centrifuge.  When 
the  number  is  still  smaller,  it  may  be  possible  to  show  their 
presence  by  guinea-pig  inoculation  though  staining  methods 
all  fail. 

The  possible  relation  that  the  number  of  bacilli  in  the 
expectoration  of  consumptives  might  bear  to  the  progress 
of  the  disease  was  investigated  by  Nuttall.f 

But  a  glance  down  the  columns  of  figures  in  the  original 
article  is  sufficient  to  show  that  the  number  of  bacilli  is  devoid 

*"Berl.  klin.  Wochenschrift,"  1898,  No.  37,  p.  809. 

f  "Bull,  of  the  Johns  Hopkins  Hospital,"  May  and  June,  1891,  n,  13. 


7i8 


Tuberculosis 


of  any  practical  interest,  as  is  only  to  be  expected  when  one 
considers  the  pathology  of  the  disease  and  remembers  that 
accident  may  cause  wide  variations  in  the  quality,  if  not  in 
the  quantity  of  the  sputum. 

Staining  the  Bacillus  in  Urine. — The  detection  of  tuber- 
cle bacilli  in  the  urine  is  sometimes  easy,  sometimes  difficult. 
The  centrifuge  should  be  used  and  the  collected  sediment 
spread  upon  the  glass.  If  there  be  no  pus  or  albumin  in 
the  urine,  it  is  necessary  to  add  a  little  white  of  egg  to 


Fig.  238. — Bacillus  tuberculosis  in  sputum,  stained  with  carbolic  fuchsin 
and  aqueous  methylene-blue.      X  1000  (Ohlmacher). 

secure  good  fixation  of  the  urinary  sediment  to  the  glass. 
The  method  of  staining  is  the  same  as  that  for  sputum. 
The  smegma  bacillus  (q.  v.)  is  apt  to  be  present  in  the  urine, 
and  the  precaution  must  be  taken  to  wash  the  specimen  with 
absolute  alcohol,  so  that  it  may  be  decolorized  and  confusion 
avoided. 

Staining  the  Bacillus  in  Feces. — It  is  difficult  to  find 
tubercle  bacilli  in  the  feces  because  of  the  relatively  small 
number  of  bacilli  and  large  bulk  of  feces. 

In  all  cases  where  the  detection  of  tubercle  bacilli  in  pus 
or  secretions  is  a  matter  of  clinical  importance,  it  must  be 


Staining  719 

remembered  that  the  quantity  of  material  examined  by  the 
staining  method  is  extremely  small,  so  that  a  few  bacilli  in 
a  relatively  large  quantity  of  matter  can  easily  escape  dis- 
covery. 

Staining  the  Bacillus  in  Sections  of  Tissue. — Ehrlich's 
Method  for  Sections. — Ehrlich's  method  must  be  recom- 
mended as  the  most  certain  and  best.  The  sections  of 
tissue,  embedded  in  paraffin,  should  be  cemented  to  the  slide 
and  then  freed  from  the  embedding  material.  They  are  then 
placed  in  the  stain  for  from  twelve  to  twenty-four  hours  and 
kept  at  a  temperature  of  37°  C.  Upon  removal  they  are 
allowed  to  lie  in  water  for  about  ten  minutes.  The  washing 
in  nitric  acid  (20  per  cent.)  which  follows  may  have  to  be 
continued  for  as  long  as  two  minutes.  Thorough  washing  in 
60  per  cent,  alcohol  follows,  after  which  the  sections  can  be 
counterstained,  washed,  dehydrated  in  96  per  cent,  and  abso- 
lute alcohol,  cleared  in  xylol,  and  mounted  in  Canada  balsam. 
Unna's  Method  for  Sections. — Unna's  method  is  as  follows : 
The  sections  are  placed  in  a  dish  of  twenty-four-hour-old, 
newly  filtered  Ehrlich's  solution,  and  allowed  to  remain 
twelve  to  twenty-four  hours  at  the  room  temperature 
or  one  to  two  hours  in  the  incubator.  From  the  stain 
they  are  placed  in  water,  where  they  remain  for  about 
ten  minutes  to  wash.  They  are  then  immersed  in  acid 
(20  per  cent,  nitric  acid)  for  about  two  minutes,  and  be- 
come greenish  black.  From  the  acid  they  are  placed 
in  absolute  alcohol  and  gently  moved  to  and  fro  until  the 
pale-blue  color  returns.  They  are  then  washed  in  three 
or  four  changes  of  clean  water  until  they  become  almost  color- 
less, and  then  removed  to  the  slide  by  means  of  a  section- 
lifter.  The  water  is  absorbed  with  filter-paper,  and  then  the 
slide  is  heated  over  a  Bunsen  burner  until  the  section  be- 
comes shining,  when  it  receives  a  drop  of  xylol  balsam  and 
a  cover-glass. 

It  is  said  that  sections  stained  in  this  manner  do  not 
fade  so  quickly  as  those  stained  by  Ehrlich's  method. 

Gram's  Method. — The  tubercle  bacillus  stains  well  by 
Gram's  method  and  by  Weigert's  modification  of  it,  but 
these  methods  are  ill  adapted  for  differentiation.  They 
should  not  be  neglected  when  no  tubercle  bacilli  are  demon- 
strable by  the  other  methods,  as  they  are  particularly  well 
adapted  to  the  demonstration  of  such  of  the  organisms  as 
may  not  be  acid-proof. 


720  Tuberculosis 

Isolation. — Piatkowski*  has  suggested  that  the  cultiva- 
tion of  the  tubercle  bacillus  and  other  "  acid-proof  "  organ- 
isms may  be  achieved  by  taking  advantage  of  their  ability  to 
resist  the  action  of  formaldehyd.  The  material  containing 
the  acid-proof  organism  is  mixed  thoroughly  with  10  c.c.  of 
water  or  bouillon,  which  receives  an  addition  of  2  or  3 
drops  of  40  per  cent,  formaldehyd  or  "  formalin."  After 
standing  from  fifteen  to  thirty  minutes  transfers  are  made  to 
appropriate  culture-media,  when  the  acid-proof  organisms 
may  develop,  the  others  having  been  destroyed  by  the  for- 
maldehyd. 

Still  further  improvement  in  the  means  by  which  the  tuber- 
cle bacilli  can  be  secured  free  from  contamination  with  other 
organisms  and  from  surrounding  unnecessary  and  undesirable 
materials,  has  accrued  from  the  use  of  antiformin.  This 
commercial  product,  patented  in  1909  by  Axel  Sjoo  andTor- 
nell,  consists  of  Javelle  water  to  which  sodium  hydrate  is 
added.  To  make  it  in  the  laboratory  one  first  makes  the 
Javelle  water  as  follows: 


K2CO3 58 

CaO(OCl)2 80 

Water, q.  s.  1000 


and  after  dissolving  the  salts  add  an  equal  volume  of  15 
per  cent,  aqueous  solution  of  caustic  soda. 

Uhlenhuth  andXylanderf  investigated  its  usefulness  and 
recommend  it  highly  for  assisting  in  manipulating  the  tuber- 
cle bacillus.  The  sputum  or  tissue  supposed  to  contain  these 
organisms  receives  an  addition  of  antiformin,  by  which  the 
tissue  elements,  the  pus  cells,  the  mucous  and  other  objec- 
tionable substances,  and  bacteria  are  quickly  dissolved,  leav- 
ing the  tubercle  bacilli  uninjured.  It  is  then  centrifugal- 
ized,  the  fluid  poured  off  and  replaced  by  sterile  water  or 
salt  solution,  and  the  bacilli  washed,  after  which  they 
are  again  centrifugalized  and  caught  at  the  bottom  of  the 
tube.  This  sediment,  rich  in  bacilli,  may  be  immediately 
transferred  to  appropriate  culture-media,  where  the  organ- 

*  " Deutsche  med.  Wochenschrift,"  June  9,  1904,  No.  23,  p.  878. 
f  "Arbeiten  a.  d.  Kaiserlichen  Gesundheilsamt,"    1909,  xxxi,   158; 
"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Referata,  1910,  xi,v,  686. 


Isolation  721 

isms  infrequently  grow  quite  well,  or  can  be  used  for  the 
inoculation  of  guinea-pigs. 

The  most  certain  method  of  obtaining  a  culture  of  the 
tubercle  bacillus  from  sputum,  pus,  etc.,  is  to  inoculate  a 
guinea-pig,  allow  artificial  tuberculosis  to  develop,  and  make 
cultures  from  one  of  the  tuberculous  lesions. 

To  make  such  an  inoculation  with  material  such  as 
sputum,  in  which  there  are  many  associated  micro-organisms 
that  may  destroy  the  guinea-pig  from  septicemia,  Koch  ad- 
vised the  following  method,  with  which  he  never  experienced 
an  unfavorable  result. 

With  a  sharp-pointed  pair  of  scissors  a  snip  about  J  cm. 
long  is  made  in  the  skin  of  the  belly- wall.  Into  this  the 
points  of  the  scissors  are  thrust,  between  the  skin  and  the 
muscles  for  at  least  i  cm.,  and  the  scissors  opened  and 
closed  so  as  to  make  a  broad  subcutaneous  pocket.  Into 
this  pocket  the  needle  of  the  hypodermic  syringe  contain- 
ing the  injection,  or  the  slender  glass  point  of  a  pipet  con- 
taining it,  is  introduced,  a  drop  of  fluid  expressed  and  gently 
rubbed  about  beneath  the  skin.  When  the  inoculating  in- 
strument is  withdrawn,  the  mouth  of  the  pocket  is  left  open. 
A  slight  suppuration  usually  occurs  and  carries  out  the  or- 
ganisms of  wound  infection,  while  the  tubercle  bacilli  are 
detained  and  carried  to  the  inguinal  nodes,  which  usually  en- 
large during  the  first  ten  days.  The  guinea-pigs  usually  die 
about  the  twenty-first  day  after  infection. 

The  guinea-pig  is  permitted  to  live  until  examination  shows 
the  inguinal  glands  are  well  enlarged,  and  toward  the  middle 
of  the  third  week  is  chloroformed  to  death.  The  exterior  of 
the  body  is  then  wet  with  i :  1000  solution  of  bichlorid  of 
mercury  and  the  animal  stretched  out,  belly  up,  and  tacked 
to  a  board  or  tied  to  an  autopsy  tray.  The  skin  is  ripped  up 
and  turned  back.  The  exposed  abdominal  muscles  are  now 
washed  with  bichlorid  solution  and  a  piece  of  gauze  wrung 
out  of  the  solution  temporarily  laid  on  to  absorb  the  excess. 
With  fresh  sterile  forceps  and  scissors  the  abdominal  wall  is 
next  laid  open  and  fastened  back.  With  the  same  instru- 
ments or,  preferably,  with  fresh  sterile  instruments  the 
spleen,  which  should  be  large  and  full  of  tubercles,  is 
drawn  forward  and,  one  after  another,  bits  the  size  of  a 
pea  cut  or  torn  off  and  immediately  dropped  upon  the  sur- 
face of  appropriate  culture-media  in  appropriate  tubes.  The 
fragments  of  tissue  from  the  spleen  of  the  tuberculous 
46 


722 


Tuberculosis 


guinea-pig  are  not  crushed  or  comminuted,  but  are  simply 
laid  upon  the  undisturbed  surface  of  the  blood-serum  and 
then  incubated  for  several  weeks.  If  no  growth  is  apparent 
after  this  period,  the  bit  of  tissue  is  stirred  about  a  little 
and  the  tube  returned  to  the  incubator,  where  growth  al- 
most immediately  begins  from  bacilli  scattered  over  the 
surface  as  the  bit  of  tissue  was  moved.  As  the  appropriate 
media,  blood-serum  was  recommended  by  Koch;  glycerin 
agar-agar,  by  Roux  and  Nocard;  glycerinized  potato,  by 

Nocard;  coagulated  dogs'  blood- 
serum,  by  Smith,  or  coagulated 
egg,  by  Dorset,  may  be  men- 
tioned. The  most  certain  results 
seem  to  follow  the  employment 
of  the  dogs'  serum  and  egg  media. 
Cultivation. — Blood-serum.— 
Koch  first  achieved  artificial  cul- 
tivation of  the  tubercle  bacillus 
upon  blood-serum,  upon  which 
the  bacilli  are  first  apparent  to 
the  naked  eye  in  about  two 
weeks,  in  the  form  of  small,  dry, 
whitish  flakes,  not  unlike  frag- 
ments of  chalk.  These  slowly 
increase  in  size  at  the  edges,  and 
gradually  form  small  scale-like 
masses,  which  under  the  micro- 
scope are  found  to  consist  of  tan- 
gled masses  of  bacilli,  many  of 
which  are  in  a  condition  of  invo- 
lution. The  medium  is  so  ill 
adapted  to  the  requirements  of 
the  tubercle  bacillus  and  gives 
such  uncertain  results  that  it  is  no  longer  used. 

Glycerin  Agar-agar. — In  1887  Nocard  and  Roux*  gave  a 
great  impetus  to  investigations  upon  tuberculosis  by  the  dis- 
covery that  the  addition  of  from  4  to  8  per  cent,  of  glycerin 
to  bouillon  and  agar-agar  made  them  suitable  for  the  devel- 
opment of  the  bacillus,  and  that  a  much  more  luxuriant  de- 
velopment could  be  obtained  upon  such  media  than  upon 
blood-serum.  The  growth  upon  "glycerin  agar-agar"  re- 
sembles that  upon  blood-serum.  A  critical  study  of  the 
*  "Ann.  de  1'Inst.  Pasteur,"  1887,  No.  i. 


Fig.  239. — Bacillus  tuber- 
culosis on  "glycerin  agar- 
agar." 


Isolation 


723 


relationship  of  massive  development  and  glycerin  was  made 
by  Kimla,  Poupe,  and  Vesley,*  who  found  that  the  most  lux- 
uriant growth  occurred  when  the  culture-media  contained 
from  5  to  7  per  cent,  of  glycerin. 

Dogs'  Blood-serum. — A  very  successful  method  of  isolating 
the  tubercle  bacillus  has  been  published  by 
Smith,  f  A  dog  is  bled  from  the  femoral  ar- 
tery, the  blood  being  caught  in  a  sterile  flask, 
where  it  is  allowed  to  coagulate.  The  serum 
is  removed  with  a  sterile  pipet,  placed  in 
sterile  tubes,  and  coagulated  at  75°  to  76°  C. 
Reichel  has  found  it  advantageous  to  add 
to  each  100  c.c.  of  the  dogs'  serum  25  c.c. 
of  a  mixture  of  glycerin  i  part  and  distilled 
water  4  parts.  The  whole  is  then  carefully 
shaken  without  making  a  froth,  and  dis- 
pensed in  tubes,  10  c.c.  to  a  tube.  The 
coagulation  and  sterilization  he  effects  by 
once  heating  to  90°  C.  for  three  to  five 
hours.  At  the  Henry  Phipps  Institute  in 
Philadelphia  I  employed  this  medium  with 
thorough  satisfaction  for  the  isolation  of 
many  different  tubercle  bacilli.  Smith  pre- 
fers to  use  a  test-tube  with  a  ground  cap, 
having  a  small  tubular  aperture  at  the  end, 
instead  of  the  ordinary  test-tube  with  the 
cotton-plug.  The  purpose  of  the  ground 
glass  cap  is  to  prevent  the  contents  of  the 
tube  from  drying  during  the  necessarily  long 
period  of  incubation;  that  of  the  tubula- 
ture,  to  permit  the  air  in  the  tubes  to  enter 
and  exit  during  the  contraction  and  expan- 
sion resulting  from  the  heating  incidental 
to  sterilization. 

To  the  same  end  the  ventilators  of  the 
incubator  are  closed,  and  a  large  evapo- 
rating dish  filled  with  water  is  stood  inside, 
so  that  the  atmosphere  may  be  constantly 
saturated  with  moisture 

Egg  Media. — DorsetJ   recommends   the   isolation  of  the 

*  "Revue  de  la  Tuberculose,"  1898,  vi,  p.  25. 

t  "  Transactions  of  the  Association  of  American  Physicians,"  1898, 
vol.  xm,  p.  417. 

I  "American  Medicine,"  1902,  vol.  in,  p.  555. 


Fig.  240. — Glass 
capped  culture- 
tube  used  by 
Theobald  Smith 
for  the  isolation 
of  the  tubercle 
bacillus. 


724 


Tuberculosis 


tubercle  bacillus  upon  an  egg  medium,  which  has  the  advan- 
tage of  being  cheap  and  easily  pre- 
pared, while  eggs  are  always  at  hand, 
and  can  be  made  into  an  appropriate 
medium  in  an  hour  or  two.  He  also 
claims  that  the  chemic  composition 
of  the  eggs  makes  them  particularly 
adapted  for  the  purpose.  The  me- 
dium is  prepared  by  carefully  open- 
ing the  egg  and  dropping  its  contents 
into  a  wide-mouth  sterile  receptacle. 
The  yolk  is  broken  with  a  sterile 
wire  and  thoroughly  mixed  with  the 
white  by  gentle  shaking.  The  mix- 
ture is  then  poured  into  sterile  tubes, 
about  10  c.c.  in  each,  inclined  in  a 
blood-serum  sterilizer,  and  sterilized 
and  coagulated  at  70°  C.  for  two 
days,  the  temperature  being  main- 
tained for  four  or  five  hours  each 
day.  The  medium  appears  yellow- 
ish and  is  usually  dry,  so  that  before 
using  it  is  well  to  use  a  few  drops  of 
water  to  make  conditions  appropri- 
ate for  the  growth  of  the  tubercle 
bacillus. 

Potato. — Pawlowski*  was  able  to 
isolate  the  bacillus  upon  potato. 
Sander  found  that  it  could  be 
readily  grown  upon  various  vege- 
table compounds,  especially  upon 
acid  potato  mixed  with  glycerin. 
Rosenauf  has  shown  that  it  can 
grow  upon  almost  any  cooked  and 
glycerinized  vegetable  tissue.  Ac- 
cording to  French  writers,  the  viru- 
lence of  the  bacillus  is  not  dimin- 
ished when  it  grows  upon  potato. 
It  has  also  been  said  that  the  con- 
tinued cultivation  of  the  tubercle 
bacillus  upon  culture-media  lessens 


Fig.  241. — Bacillus  tu- 
berculosis; glycerin  agar- 
agar  culture,  several 
months  old  (Curtis). 


'  ''Ann.  de  1'Inst.  Pasteur,"  1888,  t.  vi. 
t  "  Jour.  Amer.  Med.  Assoc.,"  1902. 


Isolation  725 

its  parasitic  nature,  so  that  in  the  course  of  time  it  can  be 
induced  to  grow  feebly  upon  the  ordinary  agar-agar,  and  that 
prolonged  cultivation  destroys  its  virulence. 

Animal  Tissues. — Frugoni*  recommends  that  the  tubercle 
bacillus  be  isolated  and  cultivated  upon  animal  tissue  and 
organs  used  as  culture-media.  He  especially  recommends 
rabbit's  lung  and  dog's  lung  for  the  purpose.  The  tissues 
are  first  cooked  in  a  steam  sterilizer,  then  cut  into  prisms, 
placed  in  a  Roux  tube,  an  addition  .of  6  to  8  per  cent, 
glycerin- water  added,  so  as  to  bathe  the  lower  part  of  the  tis- 


Fig.  242. — Bacillus  tuberculosis;  adhesion  cover-glass  preparation 
from  a  fourteen-day-old  blood-serum  culture.  X  100  (Frankel  and 
Pfeiffer). 

sue  and  keep  it  moist,  and  the  whole  then  sterilized  in  the 
autoclave. 

The  organisms  are  planted  upon  the  tissue,  the  top  of  the 
tube  closed  with  a  rubber  cap,  and  the  culture  placed  in  the 
thermostat.  The  tubercle  bacilli  grow  quickly  and  luxuriantly. 

Bouillon. — Upon  bouillon  to  which  6  per  cent,  of  glycerin 
has  been  added  the  bacillus  grows  well,  provided  the  trans- 
planted material  be  in  a  condition  to  float.  The  organism 
being  purely  aerobic,  grows  only  at  the  surface,  where  a 
much  wrinkled,  creamy  white,  brittle  pellicle  forms. 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  I.  Abl.  Orig.,  1910,  un,  553. 


726  Tuberculosis 

Non-albuminous  Media. — Instead  of  requiring  the  most 
concentrated  albuminous  media,  as  was  once  supposed, 
Proskauer  and  Beck*  have  shown  that  the  organism  can  be 
made  to  grow  in  non-albuminous  media  containing  asparagin, 
and  that  it  can  even  be  induced  to  grow  upon  a  mixture  of 
commercial  ammonium  carbonate,  0.35  per  cent.;  primary 
potassium  phosphate,  0.15  per  cent.;  magnesium  sulphate, 
0.25  per  cent.;  glycerin,  1.5  per  cent.  Tuberculin  was  pro- 
duced in  this  mixture. 

Gelatin. — The  tubercle  bacillus  can  be  grown  in  gelatin 
to  which  glycerin  has  been  added,  but  as  its  development 
takes  place  only  at  37°  to  38°  C.,  a  temperature  at  which 
gelatin  is  always  liquid,  its  use  for  the  purpose  has  no 
advantages. 


Fig.  243. — Bacillus  tuberculosis:  a,  Source,  human;  b,  source,  bovine. 
Mature  colonies  on  glycerin-agar.  Actual  size  (Swithinbank  and 
Newman) . 

Appearance  of  the  Cultures. — Irrespective  of  the  media 
upon  which  they  are  grown,  cultures  of  the  tubercle  bacil- 
lus present  certain  characteristics  which  serve  to  separate 
them  from  the  majority  of  other  organisms,  though  insuffi- 
cient to  enable  one  to  certainly  recognize  them. 

The  bacterial  masses  make  their  appearance  very  slowly. 
As  a  rule  very  little  growth  can  be  observed  at  the  end  of  a 
week,  and  sometimes  a  month  must  elapse  before  the  cul- 
tures can  be  described  as  well  grown. 

They  usually  develop  more  rapidly  upon  fluid  than  upon 
solid  media.  The  growth  is  invariably  and  purely  aerobic, 

*  "Zeitschrift  fur  Hygiene,"  Aug.  10,  1894,  xvm,  No.  i. 


Pathogenesis  727 

and  the  surface  growth  formed  upon  liquids  closely  resembles 
that  upon  solids. 

The  growth  is  dry  and  lusterless,  coarsely  granular, 
wrinkled,  slightly  yellowish,  and  does  not  penetrate  into  the 
substance  of  the  culture-medium.  It  sometimes  extends 
over  the  surface  of  the  medium  and  spreads  out  upon  the 
contiguous  surface  of  moist  glass. 

When  the  medium  is  moist,  the  bacterial  mass  may  in 
rare  instances  be  shining  in  spots,  but  it  is  usually  lusterless. 
When  the  medium  is  dry,  it  is  apt  to  be  scaly  and  almost 
chalky  in  appearance. 

The  organism  grows  well  when  once  successfully  isolated, 
and,  when  once  accustomed  to  artificial  media,  not  only 
lives  long  (six  to  nine  months)  without  transplantation,  but 
may  be  transplanted  indefinitely  without  variation. 

Reaction. — The  tubercle  bacillus  will  grow  upon  other- 
wise appropriate  media  whether  the  reaction  be  feebly 
acid  or  feebly  alkaline.  Human  tubercle  bacilli  scarcely 
change  the  reaction  of  the  media  in  which  they  grow,  but 
bovine  bacilli  produce  a  slight  acidity. 

Relation  to  Oxygen. — The  tubercle  bacillus  requires  con- 
siderable oxygen  and,  therefore,  grows  only  upon  the  surface 
of  the  culture-media. 

Temperature  Sensitivity. — The  bacillus  is  sensitive  to 
temperature  variations,  not  growing  below  29°  C.  or  above 
42°  C.  Rosenau*  found  .that  an  exposure  to  60°  C.  for 
twenty  minutes  destroys  the  infectiousness  of  the  tubercle 
bacillus  for  guinea-pigs. 

Effect  of  Light. — It  does  not  develop  well  in  the  light, 
and  when  its  virulence  is  to  be  maintained  should  always 
be  kept  in  the  dark.  Sunlight  kills  it  in  from  a  few  minutes 
to  several  hours,  according  to  the  thickness  of  the  mass  of 
bacilli  exposed  to  its  influence. 

Pathogenesis. — Channels  of  Infection. — The  channels  by 
which  the  tubercle  bacillus  enters  the  body  are  numerous.  A 
few  cases  are  on  record  where  the  micro-organisms  have 
passed  through  the  placenta,  a  tuberculous  mother  infecting 
her  unborn  child.  It  is  not  impossible  that  the  passage  of 
bacilli  through  the  placenta  in  this  manner  causes  the  rapid 
development  of  tuberculosis  after  birth,  the  disease  having 
remained  latent  during  fetal  life,  for  Birch-Hirschfeld  has 
shown  that  fragments  of  a  fetus,  itself  showing  no  tuberculous 
*  "Hygienic  Laboratory,"  Bulletin  No.  24,  Jan.,  1908. 


728  Tuberculosis 

lesions,  but  coming  from  a  tuberculous  woman,  caused  fatal 
tuberculosis  in  guinea-pigs  into  which  they  were  inoculated. 

The  most  frequent  channel  of  infection  is  the  respiratory 
tract,  into  which  the  finely  pulverized  pulmonary  discharges 
of  consumptives  and  the  dusts  of  infected  rooms  and  streets 
enter.  Fliigge,  Laschtschenko,  Heyman-Sticher,  and  Be- 
ninde*  found  that  the  greatest  danger  of  infection  was 
from  the  atomized  secretions,  discharged  during  cough,  from 
the  tuberculous  respiratory  apparatus.  Nearly  every  one 
discharges  finely  pulverized  secretions  during  coughing  and 
sneezing,  as  can  easily  be  determined  by  holding  a  mirror 
before  the  face  at  the  time.  Even  though  discharged  by 
consumptives,  these  atoms  of  moisture  are  not  infectious 
except  when  tubercle  bacilli  are  present  in  the  sputum. 
Experiment  showed  that  they  usually  do  not  pass  further 
than  0.5  meter  from  the  patient,  though  occasionally  they 
may  be  driven  1.5  meters.  A  knowledge  of  these  facts 
teaches  us  that  visits  to  consumptives  should  not  be  pro- 
longed; that  no  one  should  remain  continually  in  their 
presence,  nor  habitually  sit  within  2  meters  of  them;  also 
that  patients  should  always  hold  a  handkerchief  before 
the  face  while  coughing.  The  rooms  occupied  by  con- 
sumptives should  also  be  frequently  washed  with  a  dis- 
infecting solution. 

Probably  all  of  us  at  some  time  in  our  lives  inhale  living 
virulent  tubercle  bacilli,  yet  not  all  suffer  from  tubercu- 
losis. Personal  variations  in  predisposition  seem  to  account 
in  part  for  this,  as  it  has  been  shown  that  without  the 
formation  of  tubercles  virulent  bacilli  may  sometimes  be 
present  for  considerable  lengths  of  time  in  the  bronchial 
lymphatic  glands — the  dumping-ground  of  the  pulmonary 
phagocytes. 

In  order  that  infection  shall  occur,  it  does  not  seem 
necessary  that  the  least  abrasion  or  laceration  shall  exist 
in  the  mucous  lining  of  the  respiratory  tract. 

Infection  also  commonly  takes  place  through  the  gastro- 
intestinal tract  from  infected  food.  At  present  evidence 
points  to  danger  from  the  presence  of  tubercle  bacilli  in  the 
milk  of  cattle  affected  with  tuberculosis.  It  does  not  seem 
necessary  that  tuberculous  ulcers  shall  be  present  in  the 
udders;  indeed,  the  bacilli  have  been  demonstrated  in 

*  " Zeitschrif t  fur  Hygiene,"  etc.,  Bd.  xxx,  pp.  107,  125,  139,  163, 
193- 


Pathogenesis  729 

considerable  numbers  in  milk  from  udders  without  tuber- 
culous lesions  discoverable  to  the  naked  eye. 

The  meat  from  tuberculous  animals  is  less  dangerous  than 
the  milk,  because  it  is  nearly  always  cooked  before  being 
eaten,  while  the  milk  is  generally  consumed  in  the  raw  state. 

The  ingested  bacilli  may  enter  the  tonsils  and  be  carried 
to  the  cervical  lymph-glands,  but  seem  more  commonly 
to  reach  the  intestine,  from  which  they  enter  the  lymphatics, 
sometimes  to  produce  lesions  immediately  beneath  the 
mucous  membrane,  and  lead  to  the  later  formation  of 
ulcers;  but  usually  to  invade  the  more  distant  mesenteric 
lymphatic  glands.  Nicolas  and  Descos*  and  Ravenel  f  found 
that  when  fasting  dogs  were  fed  upon  soup  containing 
large  quantities  of  tubercle  bacilli,  they  were  able  to  dis- 
cover the  bacilli  a  few  hours  afterward  in  the  contents 
of  the  thoracic  duct.  The  thoracic  duct  is  sometimes 
affected,  and  from  such  a  lesion  it  is  easy  to  understand 
the  development  of  general  miliary  tuberculosis  through 
systemic  distribution  of  bacilli  thrown  into  the  circulation. 
The  occasional  absorption  of  tubercle  bacilli  by  the  lacteals, 
and  their  immediate  entrance  into  the  systemic  circulation 
and  subsequent  deposition  in  the  brain,  bones,  joints,  etc., 
are  supposed  to  explain  primary  lesions  of  these  tissues. 

Kochf  believes  that  human  beings  are  infected  only  by 
bacilli  from  other  human  beings,  and  his  paper  upon  this 
subject  has  stimulated  extensive  experimentation  on  the 
problem.  Most  authorities  believe  both  human  and  bo- 
vine bacilli  to  be  equally  infectious  for  man.  Behring§ 
believes  that  nearly  all  children  become  infected  by  ingest- 
ing tubercle  bacilli  in  milk,  though  a  certain  predisposition 
is  necessary  before  the  disease  can  develop.  Baumgarten 
believes  that  all  children  harbor  bacilli  taken  in  the  food, 
but  that  the  disease  does  not  develop  until  a  certain  sus- 
ceptibility occurs. 

Infection  also  occasionally  takes  place  through  the  sexual 
apparatus.  In  sexual  intercourse  tubercle  bacilli  from 
tuberculous  testicles  can  enter  the  female  organs,  with 
resulting  bacillary  implantation.  Sexual  infections  are 

'  "Jour,  de  Phys.  et  Path,  gen.,"  1902,  iv,  910. 
t  "Jour.  Med.  Research,"  x,  p.  460,  1904. 

t  "  International  Congress  on  Tuberculosis,"  London,  1901,  and 
Washington,  1908. 

§  "Deutsche  med.  Wochenschrift,"  1903,  No.  39. 


730  Tuberculosis 

usually  from  the  male  to  the  female,  primary  tuberculosis  of 
the  testicle  being  more  common  than  of  the  uterus  or  ovaries. 

Wounds  are  also  occasional  avenues  of  entrance  for 
tubercle  bacilli.  Anatomic  tubercles  are  not  uncommon 
upon  the  hands  of  anatomists  and  pathologists,  most  of 
these  growths  being  tuberculous  in  nature.  Such  dermal 
lesions  usually  contain  few  bacilli. 

Lesions. — The  macroscopic  lesions  of  tuberculosis  are  too 
familiar  to  require  a  description  of  any  considerable  length. 
They  consist  of  nodules,  or  collections  of  nodules,  called 
tubercles,  irregularly  scattered  through  the  tissues,  which 
are  more  or  less  disorganized  by  their  presence  and  retro- 
gressive changes. 

When  tubercle  bacilli  are  introduced  beneath  the  skin  of 
a  guinea-pig,  the  animal  shows  no  sign  of  disease  for  a 
week  or  two,  then  begins  to  lose  appetite,  and  gradually 
diminishes  in  flesh  and  weight.  Examination  usually  shows 
a  nodule  at  the  point  of  inoculation  and  enlargement  of  the 
neighboring  lymphatic  glands.  The  atrophy  increases,  the 
animal  shows  a  febrile  reaction,  and  dies  at  the  end  of  a 
period  of  time  varying  from  three  to  six  weeks.  Post- 
mortem examination  usually  shows  a  cluster  of  tubercles 
at  the  point  of  inoculation,  tuberculous  enlargement  of 
lymphatic  glands  both  near  and  remote  from  the  primary 
lesion,  and  a  widespread  tuberculous  invasion  of  the  lungs, 
liver,  kidneys,  peritoneum,  and  other  organs.  Tubercle 
bacilli  are  demonstrable  in  immense  numbers  in  all  the 
invaded  tissues.  The  disease  in  the  guinea-pig  is  usually 
more  widespread  than  in  other  animals  because  of  its  greater 
susceptibility,  and  the  death  of  the  animal  occurs  more 
rapidly  for  the  same  reason.  Intraperitoneal  injection  of 
tubercle  bacilli  in  guinea-pigs  causes  a  still  more  rapid  dis- 
ease, accompanied  by  widespread  lesions  of  the  abdominal 
organs.  The  animals  die  in  from  three  to  four  weeks.  In 
rabbits  the  disease  runs  a  longer  course  with  similar  lesions. 
In  cattle  and  sheep  the  infection  is  commonly  first  seen  in 
the  alimentary  apparatus  and  associated  organs,  and  may 
be  limited  to  them  though  primary  pulmonary  disease  also 
occurs.  In  man  the  disease  is  chiefly  pulmonary,  though 
gastro-intestinal  and  general  miliary  tuberculosis  are  com- 
mon. The  development  of  the  lesions  in  whatever  tissue  or 
animal  always  depends  upon  the  distribution  of  the  bacilli 
by  the  lymph  or  the  blood. 


Lesions  731 

The  experiments  of  Koch,  Prudden,  and  Hodenpyl,*  and 
others  have  shown  that  when  dead  tubercle  bacilli  are  in- 
jected into  the  subcutaneous  tissues  of  rabbits,  small  local 
abscesses  develop  in  the  course  of  a  couple  of  weeks,  show- 
ing that  the  tubercle  bacilli  possess  chemotactic  properties. 
These  chemotactic  properties '  seem  to  depend  upon  some 
other  irritant  than  that  by  which  the  chief  lesions  of  tuber- 
culosis are  caused.  When  the  dead  tubercle  bacilli,  instead 
of  being  injected  en  masse  into  the  areolar  tissue,  are  intro- 
duced by  intravenous  injection  and  disseminate  themselves 
singly  or  in  small  groups,  the  result  is  quite  different,  and 
the  lesions  closely  resemble  those  caused  by  the  living  or- 
ganisms. 

Baumgarten,  whose  researches  were  made  upon  the  iris, 
found  that  the  first  irritation  caused  by  the  bacillus  is 
followed  by  multiplication  of  the  fixed  connective-tissue 
cells  of  the  part.  The  cells  increase  in  number  by  karyo- 
kinesis,  and  form  a  minute  cellular  collection  or  primitive 
tubercle.  Such  leukocytes  as  occur  are  of  the  small  mono- 
nuclear  variety,  are  of  secondary  importance,  appear  later, 
and  are  no  doubt  attracted  both  by  the  chemotactic  sub- 
stance shown  by  Prudden  and  Hodenpyl  to  exist  in  the 
bodies  of  the  dead  bacilli  and  by  the  necrotic  changes  already 
affecting  the  tissue  in  which  the  tubercle  occurs.  For  rea- 
sons not  understood,  the  number  of  lymphocytes  varies  con- 
siderably in  different  cases.  Sometimes  there  will  be  enough 
to  justify  the  name  "  tuberculous  abscess  ";  sometimes  they 
will  be  completely  absent. 

The  essential  toxic  substance  of  the  bacillus  does  not 
cause  the  chemotaxis,  for  when  the  lymphocytes  are  absent 
the  characteristic  coagulation-necrosis  persists. 

The  group  of  epithelioid  cells  and  lymphocytes  constituting 
the  primitive  tubercle  scarcely  reaches  visible  proportions 
before  coagulation-necrosis  begins.  The  cytoplasm  of  the 
cells  takes  on  a  hyaline  character,  and  appears  to  become 
abnormally  viscid,  contiguous  cells  tending  to  fuse.  The 
chromatin  of  the  nuclei  becomes  dissolved  in  the  nuclear 
juice  and  gives  a  pale  but  homogeneous  appearance  to  the 
stained  nuclei.  Sometimes  this  nuclear  change  is  only 
observed  very  late.  There  is  little  karyorrhexis.  As  the 
necrosis  advances,  some  of  the  cells  flow  together  and  form 
large  protoplasmic  masses — giant-cells — which  contain  as 
*  "New  York  Med.  Jour.j"  June  6-20,  1891. 


732 


Tuberculosis 


many  nuclei  as  there  were  component  cells.  It  may  be 
that  the  nuclei  of  the  giant-cells  multiply  by  karyokinesis 
after  the  protoplasmic  coalescence,  but  only  one  observer, 
Baumgarten,  has  found  signs  of  this  in  giant-cells. 

Different  writers  hold  varying  opinions  concerning  the 
formation  and  office  of  the  giant  cells.  Thus,  while  I*  re- 
gard them  as  degenerative  formations,  and  unimportant 


•*•:< 

A>       •,/vV/U* 


/: a 


:im 


Fig.  244. — Miliary  tubercle  of  the  testicle:  a,  Zone  of  epithelioid  cells 
and  leukocytes;  b,  area  of  coagulation-necrosis;  c,  giant  cell  with  its 
processes;  peripherally  arranged  nuclei  and  necrotic  center;  d,  semi- 
niferous tubule  (Cameron,  in  "International  Text-book  of  Surgery"). 


entities,  there  are  many  who  believe,  with  Metchnikoif, 
that  they  are  enormous  phagocytes.  Hektoenf  believes 
that  they  are  active  bodies  from  which  cells  split  off. 

Giant  cells  are  not  always  formed  in  tubercles,  as  the 

*  "International  Medical  Magazine,"  vol.  i,  No.  10,  1892;  vol.  m, 
No.  2,  1894. 

f  "Journal  of  Experimental  Medicine,"  vol.  in,  1898,  p.  21. 


Lesions  733 

necrotic  changes  are  sometimes  so  rapid  and  widespread 
as  to  convert  the  whole  into  a  mass  of  unrecognizable  frag- 
ments. 

Tubercles  are  constantly  avascular — i.  e.,  in  them  no 
new  capillary  blood-vessels  form,  as  in  other  inflammatory 
tissues — and  the  coagulation-necrosis  soon  destroys  pre- 
existing capillaries;  the  avascularity  may  be  a  factor  in  the 
necrosis  of  the  larger  tuberculous  masses,  though  probably 
playing  no  important  part  in  the  degeneration  of  the  small 
tubercles,  which  is  purely  toxic. 

The  minute  primitive  tubercle  was  first  called  a  miliary 
tubercle,  and  small  aggregations  of  these,  "crude  tubercles," 
by  Laennec.  Tubercles  may  be  developed  in  any  tissue 
and  in  any  organ.  In  whatever  situation  they  occur,  the 
component  cells  are  either  pushed  aside  or  included  in  the 
lesion.  In  miliary  tuberculosis  of  the  kidney  it  is  not  un- 
usual to  find  a  tubercle  including  a  glomerule,  and  resolving 
its  component  capillaries  and  epithelium  into  necrotic  frag- 
ments. In  this  way  the  tissues  become  disorganized  and 
disintegrated. 

As  almost  all  tissues  contain  a  supporting  connective- 
tissue  framework,  its  fibers  must  be  embodied  in  the  new 
growth.  These  possess  little  vitality,  but  are  more  resistant 
than  the  cells,  and,  after  the  cells  of  a  tubercle  have  been 
destroyed,  may  be  distinctly  visible  among  the  granules, 
giving  the  tubercle  a  reticulated  appearance. 

As  a  rule,  tubercles  progressively  increase  in  size  by  the 
invasion  of  fresh  tissue.  The  tubercle  bacillus  does  not 
seem  to  find  the  necrotic  centers  of  the  tubercles  adapted 
to  its  growth,  and  most  of  the  bacilli  are  usually  observed 
at  the  edges,  among  the  healthy  cells,  where  the  nutri- 
tion is  good.  From  this  position  they  are  occasionally 
picked  up  by  leukocytes  and  transported  through  the 
lymph-spaces,  until  the  phagocyte  falls  a  prey  to  its  pris- 
oner, dies,  and  sows  the  seed  of  a  new  tubercle.  However, 
for  the  spread  of  tubercle  bacilli  from  place  to  place  phago- 
cytes may  not  always  be  necessary,  for  the  bacilli  can 
probably  be  transported  by  streams  of  lymph.  It  is  by 
the  steady  advance  in  necrosis  and  consolidation  that  the 
tissues  invaded  are  destroyed,  becoming  cheesy  and  crumbly 
and  forming  necrotic  masses  which,  in  the  lungs,  gradually 
crumble  away,  the  detritus  escaping  through  the  air-tubes, 
thus  forming  cavities.  From  the  beginning  of  pulmonary 


734 


Tuberculosis 


tuberculosis  the  process  of  destruction  is  greatly  accelerated 
by  inspired  saprophytic  bacteria  that  live  in  the  necrotic 


Fig.  245. — Tuberculosis  of  the  lung:  the  upper  lobe  shows  advanced 
cheesy  consolidation  with  cavity-formation,  bronchiectasis,  and  fibroid 
changes;  the  lower  lobe  retains  its  spongy  texture,  but  is  occupied  by 
numerous  miliary  tubercles. 

tissue.     The  patient  also  suffers  from  secondary  infections, 
especially  by  the  streptococcus  and  pneumococcus. 


Virulence  735 

Most  cases  of  tuberculosis  steadily  advance,  but  a  certain 
number  may  recover. 

About  the  center  of  a  typical  tubercle  there  is  a  zone  of  re- 
action in  which  the  reparative  tendency  of  the  tissue  is 
usually  but  slightly  outweighed  by  the  invasive  power  of  the 
bacilli.  If  the  vital  condition  of  the  individual  becomes  so 
changed  that  the  invasive  activity  of  the  bacilli  is  checked 
or  their  death  brought  about,  the  tubercle  begins  to  cica- 
trize, and  becomes  surrounded  by  a  zone  of  newly  formed 
contracting  fibrillar  tissue,  by  which  it  is  circumscribed 
and  isolated.  This  constitutes  recovery  from  tuberculosis. 
Sometimes  the  process  of  repair  is  accomplished  without 
the  destruction  of  the  bacilli,  which  are  incarcerated  and 
retained.  Such  a  condition  is  called  latent  tuberculosis,  and 
may  at  a  future  time  be  the  starting-point  of  a  new  in- 
fection. 

Virulence. — The  virulence  of  tubercle  bacilli  varies 
considerably  according  to  the  sources  from  which  they  are 
obtained.  Bacilli  from  different  cases  are  of  different 
degrees  of  virulence,  and  bacilli  from  different  animals  vary 
still  more.  Lartigau,*  in  an  instructive  paper  upon  "  Varia- 
tion in  Virulence  of  the  Bacillus  Tuberculosis  in  Man," 
found  much  variation  among  bacilli  secured  from  the  lesions 
of  human  tuberculosis.  The  virulence  was  tested  by  em- 
ploying cultures  only  for  inoculation,  and  taking  of  each 
bacillary  mass  exactly  5  mg.  by  weight,  suspending  it  in  5  c.c. 
of  an  indifferent  fluid  until  the  density  was  uniform  and 
the  microscope  showed  no  clumps,  and  injecting  into  rab- 
bits and  guinea-pigs,  pairs  of  animals  being  injected  in 
the  same  manner,  with  the  same  material,  at  the  same 
time,  and  being  subsequently  kept  under  similar  conditions. 
The  occurrence  of  tuberculosis  in  the  inoculated  animals 
was  decided  by  both  macroscopic  and  microscopic  tests. 

Lartigau  found  that  human  tubercle  bacilli  from  different 
sources  produced  varying  degrees  of  tuberculosis  in  animals ; 
that  the  injection  of  the  same  culture  in  different  amounts 
produces  different  results;  that  the  extent  and  rapidity  of 
development  usually  corresponds  to  the  virulence  of  the 
culture;  that  doses  of  i  mg.  of  a  very  virulent  culture  may 
induce  general  tuberculosis  in  rabbits  in  a  very  short  time; 
that  20  mg.  of  a  bacillus  of  low  virulence  may  fail  to  pro- 

c  "Journal  of  Medical  Research,"  vol.  vi,  No.  i;  N.  S.,  vol.  i,  No.  i, 
p.  156,  July,  1901. 


736  Tuberculosis 

duce  any  lesion  in  rabbits  or  guinea-pigs ;  that  no  morphologic 
relationship  could  be  observed  between  the  bacilli  and  their 
virulence;  that  highly  virulent  bacilli  grew  scantily  on 
culture-media  and  were  short  lived;  that  bacilli  of  widely 
different  virulence  may  be  present  in  any  one  of  the  various 
human  tuberculous  lesions ;  that  in  scrofulous  lymphadenitis 
the  bacilli  are  usually  of  low  virulence;  the  bacilli  in  pul- 
monary tuberculosis  with  ulcer ation  are  of  feeble  virulence, 
those  of  miliary  tuberculosis  of  very  great  virulence;  that 
the  so-called  "  healed  tubercles  "  of  the  lung  may  contain 
virulent  or  attenuated  bacilli;  that  individuals  suffering 
from  infection  with  a  bacillus  of  a  low  grade  of  virulence 
may  be  again  infected  with  extremely  virulent  tubercle 
bacilli;  that  chronic  tuberculosis  of  the  bones  may  contain 
bacilli  of  high  or  low  virulence,  and  that  variations  in 
virulence  among  human  tubercle  bacilli  may  possibly  some- 
times depend,  like  many  other  qualities  among  tubercle 
bacilli,  on  peculiarities  inherited  through  serial  trans- 
missions in  other  than  human  hosts. 

Chemistry  of  the  Tubercle  Bacillus. — Klebs*  found 
that  the  tubercle  bacillus  contains  two  fatty  bodies,  one 
of  which,  having  a  reddish  color  and  melting  at  42°  C., 
can  be  extracted  with  ether.  It  forms  about  20  per  cent, 
by  weight  of  the  bacillary  substance.  The  other  is  in- 
soluble in  ether,  but  soluble  in  benzole,  with  which  it  can 
be  extracted.  It  melts  at  about  50°  C.  and  constitutes 
1.14  per  cent,  of  the  bacillary  substance.  After  removing 
these  fatty  bodies  the  bacilli  fail  to  resist  the  decolorant 
action  of  acids  when  stained  by  ordinary  methods,  so  that 
it  seems  probable  that  their  acid-resisting  power  depends 
upon  them. 

De  Schweinitzf  showed  that  it  was  possible  to  extract 
from  the  tubercle  bacillus  an  acid  closely  resembling,  if 
not  identical  with,  teraconic  acid.  It  melts  at  161°  to  164°  C. 
and  is  soluble  in  ether,  water,  and  alcohol.  He  thinks  the 
necrotic  changes  caused  by  the  organism  depend  upon  it. 

RuppelJ  believes  that  three  different  fatty  substances 
are  present  in  the  tubercle  bacillus,  making  up  from  8  to 
26  per  cent,  by  weight.  The  first  can  be  extracted  with 

*  "Centralbl.  f.  Bakt.,"  1896,  xx,  p.  488. 

t  "Trans.  Assoc.  of  Amer.  Phys.,"  1897;  "Centralbl.  f.  Bakt.,"  etc., 
Sept.  15,  1897,  Bd.  xxn,  p.  200. 

J  "Zeitschrift  fur  physiol.  Chemie,"  1899,  xxvi. 


Toxic  Products  737 

cold  alcohol,  the  second  with  hot  alcohol,  the  third  with 
ether.  In  addition  to  the  fatty  substance  Ruppel  also 
found  what  he  believes  to  be  a  protamin,  and  calls  tuber - 
culosamin.  It  seems  to  be  combined  with  nucleinic  acid, 
and,  indeed,  from  it  he  isolated  an  acid  for  which  he  proposes 
the  name  tuber culinic  acid. 

Behring*  found  that  this  acid  contained  a  histon-like 
body  whose  removal  left  chemically  pure  tuber  culinic  acid. 
One  gram  of  this  acid  is  capable  of  killing  a  6oo-gram  guinea- 
pig  when  administered  beneath  the  skin.  One  gram  is 
fatal  to  90,000  grams  of  guinea-pig  when  introduced  into 
the  brain.  If  injected  into  tuberculous  guinea-pigs  it  is 
much  more  fatal,  i  gram  destroying  60,000  when  injected 
subcutaneously  and  40,000,000  when  injected  into  the 
brain. 

Levenef  also  found  free  and  combined  nucleinic  acid 
varying  in  phosphorus  content  from  6.58  to  13.19  per  cent. 
He  also  found  a  glycogen-like  substance  that  reduced 
Fehling's  solution  when  heated  with  a  mineral  acid. 

Toxic  Products. — In  1890  Koch  I  announced  some  ob- 
servations upon  the  toxic  products  of  the  tubercle  bacillus 
and  their  relation  to  the  diagnosis  and  treatment  of  tubercu- 
losis, which  at  once  aroused  an  enormous  though  transitory 
enthusiasm.  The  observations  are,  however,  of  great 
importance.  Koch  found  that  when  guinea-pigs  are 
inoculated  with  tubercle  bacilli,  the  wound  ordinarily  heals 
readily,  and  soon  all  signs  of  local  disturbance  other  than 
enlargement  of  the  lymphatic  glands  of  the  neighborhood 
disappear.  In  about  two  weeks,  however,  there  appears, 
at  the  point  of  inoculation  a  slight  induration,  which  develops 
into  a  hard, nodule,  ulcerates,  and  remains  until  the  death 
of  the  animal.  If,  however,  in  a  short  time  the  animals  be 
reinoculated,  the  course  of  the  local  lesion  is  changed,  and, 
instead  of  healing,  the  wound  and  the  tissue  surrounding  it 
assume  a  dark  color,  become  obviously  necrotic,  and  ulti- 
mately slough  away,  leaving  an  ulcer  which  rapidly  and 
permanently  heals  without  enlargement  of  the  lymph- 
glands. 

This  observation  was  made  by  injecting  cultures  of  the 
living  bacillus,  but  Koch  observed  that  the  same  changes 

*"  Berliner  klin.  Wochenschrift,"  xxxvi. 
t  "  Jour,  of  Med.  Research,"  I,  1901. 
J  "Deutsche  med.  Wochenschrift,"  1891,  No.  343. 
47 


738  Tuberculosis 

also  occur  when  the  secondary  inoculation  is  made  with 
killed  cultures  of  the  bacilli. 

It  was  also  observed  that  if  the  material  used  for  the 
secondary  injections  was  not  too  concentrated  and  the 
injections  not  too  often  repeated  (only  every  six  to  forty- 
eight  hours),  the  animals  treated  improved  in  condition, 
and  continued  to  live,  sometimes  (Pfuhl)  as  long  as  nineteen 
weeks. 

Tuberculin. — Koch  also  discovered  that  a  50  per  cent, 
glycerin  extract  of  cultures  of  the  tubercle  bacillus — tuber- 
culin— produced  the  same  effect  as  the  dead  cultures  orig- 
inally used,  and  announced  the  discovery  of  this  substance 
to  the  scientific  world,  in  the  hope  that  the  prolongation  of 
life  observed  to  follow  its  use  in  the  guinea-pig  might  also 
be  true  of  man. 

The  active  substance  of  the  "  tuberculin  "  seems  to  be 
an  albuminous  derivative  (bacterioprotein)  insoluble  in 
absolute  alcohol.  It  is  a  protein  substance  and  gives  all 
the  characteristic  reactions.  It  differs  from  the  toxalbumins 
in  being  able  to  resist  exposure  to  120°  C.  for  hours  without 
change.  Tuberculin  is  almost  harmless  for  healthy  animals, 
but  extremely  poisonous  for  tuberculous  animals,  its  injec- 
tion into  them  being  followed  either  by  a  violent  febrile 
reaction  or  by  death,  according  to  the  extent  of  the  dis- 
ease and  size  of  the  dose  administered. 

Preparation  of  Tuberculin. — The  preparation  of  tuberculin  is  simple. 
Flasks  made  broad  at  the  bottom  so  as  to  expose  a  considerable  sur- 
face of  the  contained  liquid  are  filled  to  a  depth  of  about  2  cm.  with 
bouillon  containing  4  to  6  per  cent,  of  glycerin,  and  preferably  made 
with  veal  instead  of  beef  infusion.  They  are  inoculated  with  pure  cul- 
tures of  the  tubercle  bacillus,  care  being  taken  that  the  bacillary  mass 
floats  upon  the  surface,  and  are  kept  in  an  incubator  at  37°  C.  In  the 
course  of  some  days  a  slight  surface  growth  becomes  apparent  about  the 
edges  of  the  floating  bacillary  mass,  which  in  the  course  of  time  develops 
into  a  firm,  coarsely  granular,  wrinkled  pellicle.  At  the  end  of  some 
weeks  development  ceases  and  the  pellicle  sinks,  a  new  growth  some- 
times occurring  from  floating  scraps  of  the  original. 

Some  bacteriologists  prefer  to  use  small  Erlenmeyer  flasks  for  the 
purpose,  but  large  flasks,  which  contain  from  500  c.c.  to  i  liter,  are  more 
convenient.  The  contents  of  a  number  of  flasks  of  well-grown  cultures 
are  poured  into  a  large  porcelain  evaporating  dish,  concentrated  over  a 
water-bath  to  one-tenth  their  volume,  and  filtered  through  a  Pasteur- 
Chamberland  filter.  This  is  crude  tuberculin. 

When  doses  of  a  fraction  of  a  cubic  centimeter  of  crude  tuberculin 
are  injected  into  tuberculous  animals,  an  inflammatory  and  febrile 
reaction  occurs.  Superficial  tuberculous  lesions  (lupus)  sometimes 
ulcerate  and  slough  away.  The  febrile  reaction  is  sufficiently  character- 
istic to  be  of  diagnostic  value,  though  tuberculin  can  only  be  used  with 
perfect  safety  as  a  diagnostic  agent  upon  the  lower  animals. 


Toxic  Products 


739 


From  the  "crude"  or  original  tuberculin  Koch  prepared  a  purified  or 
"refined"  tuberculin  by  adding  one  and  one-half  volumes  of  absolute 
alcohol,  stirring  thoroughly,  and  standing  aside  for  twenty-four  hours. 
At  the  end  of  this  time  a  flocculent  deposit  will  be  seen  at  the  bottom  of 
the  vessel.  The  supernatant  fluid  is  carefully  decanted  and  an  equal 
volume  of  60  per  cent,  alcohol  poured  into  the  vessel  for  the  purpose  of 
washing  the  precipitate,  which  is  again  permitted  to  settle,  the  fluid 
decanted,  and  the  washing  thus  repeated  several  times,  after  which  it  is 


Fig.  246. — Massive  culture  of  the  tubercle  bacillus  upon  the  surface  of 
glycerin-bouillon,  used  in  the  manufacture  of  tuberculin. 

finally  washed  in  absolute  alcohol  and  dried  in  a  vacuum  exsiccator. 
The  white  powder  thus  prepared  is  fatal  to  tuberculous  guinea-pigs  in 
doses  of  2  to  10  mg.  It  is  soluble  in  water  and  glycerin  and  gives  the  pro- 
tein reactions.  The  tuberculin  as  Koch  prepared  it  is  now  known  as 
"concentrated"  or  "Koch's  tuberculin,"  to  differentiate  it  from  the 
"diluted  tuberculin"  sometimes  sold  in  the  shops,  which  is  the  same 
thing  so  diluted  with  i  per  cent,  aqueous  carbolic  acid  solution  that  i  c.c. 
equals  a  dose.  The  dose  of  the  concentrated  tuberculin  is  0.4  to  0.5  c.c. ; 
that  of  the  diluted  tuberculin,  i  c.c. 


740  Tuberculosis 

Tuberculin  does  not  exert  the  slightest  influence  upon  the 
tubercle  bacillus,  but  acts  upon  the  tuberculous  tissue,  aug- 
menting the  poisonous  influence  upon  the  cells  surrounding 
the  bacilli,  destroying  their  vitality,  and  removing  the  condi- 
tions favorable  to  bacillary  growth,  which  for  a  time  is 
checked.  This  action  is  accompanied  by  marked  hyperemia 
of  the  perituberculous  tissue,  with  transudation  of  serum, 
softening  of  the  tuberculous  mass,  and  its  absorption  into 
the  blood,  a  marked  febrile  reaction  resulting  from  the 
intoxication. 

Virchow,  who  well  understood  the  action  of  the  tuberculin, 
soon  showed  that  as  a  diagnostic  and  therapeutic  agent  in 
man  its  use  was  attended  by  grave  dangers.  The  destroyed 
tissue  was  absorbed,  but  with  it  some  of  the  bacilli,  which, 
being  transported  to  new  tissue  areas,  could  occasion  a 
widespread  metastatic  invasion  of  the  disease.  Old  tuber- 
culous lesions  which  had  been  encapsulated  were  sometimes 
softened  and  broken  down,  and  became  renewed  sources  of 
infection  to  the  individual,  so  that,  a  short  time  after  an 
enthusiastic  reception,  tuberculin  was  placed  upon  its  proper 
footing  as  an  agent  valuable  for  diagnosis  in  veterinary 
practice,  but  dangerous  in  human  medicine,  except  in  cases 
of  lupus  and  other  external  forms  of  tuberculosis  where  the 
destroyed  tissue  could  be  readily  discharged  from  the  surface 
of  the  body. 

Many,  however,  continued  to  use  it,  and  Petruschky*  has 
reported,  with  careful  details,  22  cases  of  tuberculosis  which 
he  claims  have  been  cured  by  it. 

Recently  there  has  been  a  return  to  the  use  of  tuberculin 
for  the  diagnosis  of  tuberculosis,  it  being  claimed  that  by  the 
use  of  minute  doses,  several  times  repeated,  the  charac- 
teristic reaction  and  a  positive  diagnosis  can  be  obtained 
without  danger. 

von  Pirquetf  found  that  if  a  drop  or  two  of  Koch's  (old) 
tuberculin  is  placed  upon  the  skin  of  a  tuberculous  child, 
and  a  small  scarification  made  through  the  drop  with  a 
sterile  lancet,  a  small  papule  develops  at  the  point  of  inocula- 
tion that  is  not  unlike  a  vaccine  papule.  It  is  at  first  bright, 
later  on  dark  red,  and  remains  for  a  week.  Out  of  500  tests 
made,  the  results  were  positive  in  nearly  every  case  of 
clinical  tuberculosis.  The  most  characteristic  reactions 

*  "Berliner  klin.  Wochenschrift,"  1899,  Dec.  18-25. 
t  Ibid.,  May  20,  1907. 


Toxic  Products  741 

were  obtained  in  tuberculosis  of  the  bones  and  glands,  and 
the  method  is  recommended  chiefly  for  the  diagnosis  of 
tuberculosis  during  the  first  year  of  life.  This  method  of 
testing  is  called  the  dermotuberculin  reaction. 

A  modification  of  this  method  by  Lignieres*  is  called  by 
him  the  cutituberculin  reaction.  Lignieres  soaps  and  shaves 
the  skin  with  a  safety  razor,  avoiding  scarification,  but 
removing  the  superficial  epidermal  cells  by  scraping,  and 
then  applies  6  large  drops  of  undiluted  tuberculin,  rubbing 
the  reagent  in  with  a  pledget  of  cotton.  The  reaction 
obtained  is  purely  local  and  without  fever. 

Morrof  has  improved  upon  von  Pirquet's  method  by  using 
the  tuberculin  in  the  form  of  a  50  per  cent,  ointment  made  by 
mixing  equal  parts  of  "old  tuberculin"  and  lanolin,  which  is 
rubbed  into  the  skin  without  previous  scarification. 

HissJ  says  that  "it  is  more  simple  and  equally  efficient 
to  massage  into  the  skin  a  drop  of  undiluted  '  old  tuberculin.'  " 
•  Calmette§  suggested  the  "  ophthalmo-tuberculin  reaction," 
which  consists  of  dropping  i  drop  of  a  solution  of  prepared 
tuberculin  into  the  eye  of  the  suspect.  If  no  tuberculosis  ex- 
ists, no  reaction  follows,  but  if  the  patient  be  infected  with 
tuberculosis,  the  eye  becomes  reddened  in  a  few  hours  and 
soon  shows  all  of  the  appearances  of  a  more  or  less  pronounced 
acute  mucopurulent  inflammation  of  the  conjunctiva.  This 
attains  its  maximum  in  six  or  seven  hours,  and  entirely 
recovers  in  three  days.  It  usually  causes  the  patient  very 
little  discomfort,  but  a  number  of  patients  have  been  un- 
fortunate enough  to  suffer  from  supervening  corneal  ulcera- 
tion  and  other  destructive  lesions  of  the  eye,  so  that  the 
test  is  now  rarely  used,  having  been  superseded  by  the  dermal 
methods. 

The  method  of  preparing  the  solution  employed  by  Cal- 
mette  is  to  precipitate  the  tuberculin  with  alcohol,  dry  the 
precipitate,  and  dissolve  it  in  100  parts  of  distilled  water. 
One  or  two  drops  may  be  used.  Ordinary  tuberculin  must 
be  avoided,  as  the  glycerin  it  contains  causes  too  much 
irritation  and  masks  the  reaction. 

Priority    in    regard    to    the    theoretic   aspects   of    these 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  orig.,  XLVI,  Hft.  4,  March  10, 
1908,  p.  373. 

t  "Munch,  med.  Wochenschrift,"  1906,  p.  216. 
%  "Text-book  of  Bacteriology,"  1910,  p.  489. 
§  "La  Presse  Medicale,"  June  19,  1907. 


742  Tuberculosis 

reactions  seems  to  belong  to  Wolff-Eisner,*  who  was  the 
first  to  point  out  that  the  injection  of  all  albuminous  sub- 
stances resulted  in  hypersensitivity  instead  of  immunity 
unless  certain  precautions  were  observed.  Upon  this  ground 
Levyf  gives  him  credit  as  the  founder  of  the  method.  The 
reaction  is  undoubtedly  one  of  anaphylaxis  (q.  v.). 

KlebsJ  has  made  strong  claims  for  his  own  modifications 
of  tuberculin,  known  as  antiphthisin  and  tuber culocidin. 
According  to  the  experimental  studies  of  Trudeau  and  Bald- 
win, however,  antiphthisin  is  only  much  diluted  tuberculin, 
and  exerts  no  demonstrable  influence  upon  the  tubercle 
bacillus  in  vitro,  does  not  cure  tuberculosis  in  guinea-pigs,  and 
probably  inhibits  the  growth  of  the  tubercle  bacillus  upon 
culture-media  to  which  it  has  been  added  only  by  its  acid 
reaction.  The  preparations  are  no  longer  mentioned  in  the 
literature  except  as  having  failed  to  cure  tuberculosis. 

The  "  bouillon-filtrate  "  (Bouillon  filtre)  of  Denys§  is  a 
porcelain  filtrate  of  bouillon  culture  of  the  tubercle  bacillus 
and  corresponds  to  Koch's  original  tuberculin  before  con- 
centration, except  in  that  it  has  not  been  subjected  to 
heat. 

Tuberculin-R. — What  appears  to  be  an  important  modi- 
fication of  tuberculin  has  been  made  by  Koch,||  in  the 
TR  or  tuberculin-R. 

All  attempts  to  produce  immunity  against  the  tubercle 
bacillus  by  the  injection  of  attenuated  cultures,  whether 
dead  or  alive,  fail  because  of  the  invariable  occurrence  of 
abscesses  following  their  introduction  into  the  cellular 
tissue,  and  of  nodular  growths  in  the  lungs  succeeding  their 
injection  into  the  circulation.  It  seemed  as  if  the  fluids  of 
the  body  could  not  effect  the  solution  of  the  bacteria  and  the 
liberation  of  their  essential  toxic  and  immunizing  con- 
stituents. 

Koch,  therefore,  endeavored  to  bring  about  artificial  con- 
ditions advantageous  to  the  absorption  of  the  bacilli,  and 
for  the  purpose  tried  the  solvent  action  of  diluted  mineral 
acids  and  alkalies.  The  changes  thus  brought  about  facili- 

*"Centralbl.  f.  Bakt.  u.  Parasitenk.,"  orig.,  xxxvn,  1904. 
f  "Verein  fur  innere  Medizin  zu  Berlin,"  Dec.  16,  1907. 
t  "Die  Behandlung  der  Tuberculose  mit  Tuberculocidin,"  1892. 
§  "Acad.  royale  de  med.  de  Belgique,"  Feb.  22,  1902;  abst.  "Cen- 
tralbl.  f.  Bakt.  u.  Parasitenk.,"  Ref.,  1902,  xxxi,  p.  563. 
||  "Deutsche  med.  Wochenschrift,"  1897,  No.  14. 


Toxic  Products  743 

tated  absorption,  but  the  absorption  of  bacilli  in  this  chem- 
ically altered  condition  was  not  followed  by  immunity,  prob- 
ably because  the  chemic  composition  of  tubercle  toxin  (or 
whatever  one  may  name  the  poisonous  product  of  the 
bacillus)  was  altered  by  the  reagents. 

Tuberculin,  with  which  Koch  performed  many  experi- 
ments, was  found  to  produce  immunity  only  against  tuber- 
culin, not  against  bacillary  infection. 

Pursuing  the  idea  of  fragmenting  the  bacilli,  or  treating  them  chem- 
ically to  increase  their  solubility,  Koch  found  that  a  10  per  cent,  sodium 
hydrate  solution  yielded  an  alkaline  extract  of  the  bacillus,  which,  when 
injected  into  animals,  produced  effects  similar  to  those  following  the 
administration  of  tuberculin,  except  that  they  were  more  brief  in  dura- 
tion and  more  constant  in  result ;  but  the  disadvantage  of  abscess  for- 
mation following  the  injections  remained.  The  fluid,  when  filtered, 
possessed  the  properties  of  tuberculin. 

Mechanical  fragmentation  of  bacilli  had  been  employed  by  Klebs 
in  his  studies  of  antiphthisin  and  tuber culocidin,  and  Koch  now  used  it 
with  advantage.  He  pulverized  living,  virulent,  but  perfectly  dry 
bacilli  in  an  agate  mortar,  in  order  to  liberate  the  toxic  substance  from  its 
protecting  envelop  of  fatty  acid,  triturating  only  very  small  quantities 
of  the  bacteria  at  a  time. 

Having  thus  reduced  the  bacilli  to  fragments,  he  removed  them  from 
the  mortar,  placed  them  in  distilled  water,  washed  them,  and  collected 
them  by  centrifugation,  as  a  muddy  residuum  at  the  bottom  of  an  opal- 
escent, clear  fluid.  For  convenience  he  named  the  clear  fluid  TO;  the 
sediment,  TR.  TO  was  found  to  contain  tuberculin.  In  order  to 
separate  the  essential  poison  of  the  bacteria  as  perfectly  as  possible  from 
the  irritating  tuberculin,  the  TR  fragments  were  again  dried  perfectly, 
triturated  once  more,  re-collected  in  fresh  distilled  water,  and  recen- 
trifugated.  After  the  second  centrifugation  microscopic  examination 
showed  that  the  bacillary  fragments  had  not  yet  been  resolved  into  a 
uniform  mass,  for  when  TO  was  subjected  to  staining  with  carbol-fuchsin 
and  methylene-blue  it  was  found  to  exhibit  a  blue  reaction,  while  in  TR 
a  cloudy  violet  reaction  was  obtained. 

The  addition  of  50  per  cent,  of  glycerin  had  no  effect  upon  TO,  but 
caused  a  cloudy  white  deposit  to  be  thrown  down  from  TR.  This  last 
reaction  showed  that  TR  contained  fragments  of  the  bacilli  insoluble  in 
glycerin. 

In  making  the  TR  preparation  Koch  advises  the  use  of  a  fresh,  highly 
virulent  culture  not  too  old.  It  must  be  perfectly  dried  in  a  vacuum 
exsiccator,  and  the  trituration,  in  order  to  be  thorough,  should  not  be 
done  upon  more  than  100  mg.  of  the  bacilli  at  a  time.  A  satisfactory 
separation  of  the  TR  from  TO  is  said  to  only  occur  when  the  perfectly 
clear  TO  takes  up  at  least  50  per  cent,  of  the  solid  substance,  as  otherwise 
the  quantity  of  TO  in  the  final  preparation  is  so  great  as  to  produce  un- 
desirable reactions. 

The  fluid  is  best  preserved  by  the  addition  of  20  per  cent,  of  glycerin, 
which  does  not  injure  the  TR  and  prevents  its  decomposition. 

The  finished  fluid  contains  10  mg.  of  solid  constituents  to  the  cubic 
centimeter,  and  before  administration  should  be  diluted  with  physiologic 
salt  solution  (not  solutions  of  carbolic  acid).  When  administering  the 
remedy  to  man  the  injections  are  made  with  a  hypodermic  syringe  into 
the  tissues  of  the  back.  The  beginning  dose  is  7£7  mg.,  rapidly  increased 
to  20  mg.,  the  injections  being  made  daily. 


744  Tuberculosis 

Experiment  showed  that  TR  had  decided  immunizing 
powers.  Injected  into  tuberculous  animals  in  too  large  a  dose 
it  produces  a  reaction,  but  its  immunizing  effects  were  entirely 
independent  of  the  reaction.  Koch's  aim  in  using  this 
preparation  in  the  therapeutic  treatment  of  tuberculosis  was 
to  produce  immunity  against  the  tubercle  bacillus  without 
reactions  by  gradual  but  rapid  increase  of  the  dose.  In  so 
large  a  number  of  cases  did  Koch  produce  immunity  to 
tuberculosis  by  the  administration  of  TR,  that  he  believes 
it  proved  beyond  a  doubt  that  his  observations  are  correct. 

By  proper  administration  of  the  TR  he  was  able  to  render 
guinea-pigs  so  completely  immune  that  they  were  able 
to  withstand  inoculation  with  virulent  bacilli.  The  point 
of  inoculation  presents  no  change  when  the  remedy  is  ad- 
ministered; and  the  neighboring  lymph-glands  are  generally 
normal,  or  when  slightly  swollen  contain  no  bacilli. 

In  speaking  of  his  experiments  upon  guinea-pigs,  Koch 
says: 

"I  have,  in  general,  got  the  impression  in  these  experiments  that  full 
immunization  sets  in  two  or  three  weeks  after  the  use  of  large  doses.  A 
cure  in  tuberculous  guinea-pigs,  animals  in  which  the  disease  runs,  as  is 
well  known,  a  very  rapid  course,  may,  therefore,  take  place  only  when 
the  treatment  is  introduced  early — as  early  as  one  or  two  weeks  after  the 
infection  with  tuberculosis. 

''This  rule  avails  also  for  tuberculous  human  beings,  whose  treat- 
ment must  not  be  begun  too  late.  ...  A  patient  who  has  but  a  few 
months  to  live  cannot  expect  any  value  from  the  use  of  the  remedy,  and 
it  will  be  of  little  use  to  treat  patients  who  suffer  chiefly  from  secondary 
infection,  especially  with  the  streptococcus,  and  in  whom  the  septic 
process  has  put  the  tuberculosis  entirely  in  the  background." 

One  very  serious  objection,  first  urged  against  commer- 
cially prepared  TR  by  Trudeau  and  Baldwin,*  is  that  it  is 
possible  for  it  to  contain  unpulverized,  and  hence  still  living, 
virulent  tubercle  bacilli.  Thellingf  could  not  observe  any 
good  effect  to  result  from  the  use  of  Koch's  new  tuberculin, 
and,  like  Trudeau,  found  living,  virulent  bacilli  in  the  prepa- 
ration secured  from  Hochst.  Many  others  have  since  dis- 
covered the  same  danger.  In  the  preparation  of  the  remedy 
it  will  be  remembered  that  no  antiseptic  or  germicide  was 
added  to  the  solutions  by  which  the  effects  of  accidental  fail- 
ure to  crush  every  bacillus  could  be  overcome,  Koch  having 
specially  deprecated  such  additions  as  producing  destruc- 
tive changes  in  the  TR.  Until  this  possibility  of  danger  can 
be  removed,  and  our  confidence  that  attempts  to  cure 

*  "Medical  News,"  Aug.  28,  1897. 

t  "Centralbl.  f.  Bakt.,"  etc.,  July  5,  1902,  xxxn,  No.  i,  p.  28. 


Agglutination  745 

patients  may  not  result  in  their  infection  be  restored,  it  be- 
comes a  question  whether  TR  can  find  a  place  in  human 
medicine,  or  must  remain  an  interesting  laboratory  product. 

Baumgarten  and  Walz*  find  that  the  administration  of 
tuberculin-R  to  guinea-pigs  is  without  curative  effect. 
They  insist  that  the  results  obtained  are  like  those  of  the 
old  tuberculin;  that  "  small  doses  are  of  no  advantage, 
while  the  larger  the  doses  one  employs,  the  greater  are  the 
disadvantages  that  result  from  their  employment." 

During  his  experiments  upon  the  agglutination  of  tubercle 
bacilli,  to  be  described  below,  Kochf  found  that  animals 
injected  with  an  emulsion  of  tubercle  bacilli  showed  great 
increase  in  the  agglutinative  power  of  the  blood.  This 
led  him  to  suggest  that  a  new  preparation,  "  bacillary  emul- 
sion "  [Bazillen  emulsion]  be  investigated  for  its  immunizing 
and  curative  properties.  Many  are  still  using  it  and  some 
claim  good  results. 

It  is  almost  impossible  to  make  an  accurate  estimation 
of  the  usefulness  or  uselessness  of  therapeutic  preparations 
of  tubercle  bacilli  at  the  present  time,  not  only  because  of 
their  diversity  of  composition  and  the  enthusiasm  with 
which  many  have  been  exploited,  but  also  because  of  our 
inability  to  compare  the  results  attained  with  any  definite 
standard.  The  advantages  or  disadvantages  of  any  prepara- 
tion, therefore,  depend  upon  the  personal  opinions  of  those 
employing  them  rather  than  upon  any  demonstration  re- 
garding them — a  very  unscientific  state  of  knowledge. 

The  suggestion  of  A.  K.  Wright  that  the  administration 
of  all  such  products  should  be  governed  by  an  examination 
of  the  opsonic  power  of  the  blood,  the  remedy  being  withheld 
if  this  was  high  and  applied  if  low,  the  utmost  care  being 
taken  not  to  prolong  the  "  negative  phase,"  seemed  to  be  an 
excellent  one,  affording  the  beginning  of  a  scientific  method 
of  studying  the  disease,  but  unfortunately  it  seems  not  to 
have  been  successful  in  practice,  and  the  tedium  and  ex- 
pense of  the  examinations  makes  them  impracticable. 

Agglutination. — Arloingi  and  Courmont§  found  it  pos- 

*  "Centralbl.  f.  Bakt.  und  Parasitenk.,"  April  12,  1898,  xxm,  No. 
14,  P-  593- 

f  "Deutsche  med.  Wochenschrift,"  1901,  No.  48,  p.  829. 

%  "Congress  de  med.  int.  Montpellier,"  1898;  "Compt.  rendu  Acad. 
de  Sciences  de  Paris,"  1898,  T.  cxxvi,  pp.  1319-1321. 

§  "Compt.  rend.  Soc.  de  Biol.  de  Paris,"  1898,  No.  28,  v;  "Congr. 
pour  1'etude  de  la  Tuberculose,"  Paris,  1898. 


746  Tuberculosis 

sible  to  prepare  homogenized  cultures  of  the  tubercle  bacil- 
lus, and  saw  them  agglutinated  by  the  serum  of  immu- 
nized animals  and  by  the  serum  of  tuberculous  patients. 
The  subject  was  investigated  by  Koch,*  who  carefully 
reviews  the  details  of  technic  and  investigates  the  method, 
which,  he  concludes,  is  valueless  for  the  diagnosis  of  human 
infection,  though  a  good  guide  to  the  extent  of  immunization 
achieved  by  the  therapeutic  administration  of  tuber culin-R. 
Thellingf  has  also  shown  the  reaction  to  be  too  irregular 
to  be  of  practical  diagnostic  importance. 

The  technic  of  the  agglutination  test  as  given  by  KochJ 
is  as  follows : 

Any  culture  of  the  tubercle  bacillus  can  be  made  useful  by  the  fol- 
lowing treatment:  Collect  the  bacillary  masses  upon  a  filter-paper 
and  press  between  layers  of  filter-paper  to  remove  the  fluid.  Weigh 
out,  say,  0.2  gm.  of  the  solid  mass  and  rub  it  in  an  agate  mortar,  adding, 
drop  by  drop,  a  ^V  normal  sodium  hydroxid  solution  until  the  proportion 
of  i  part  of  the  culture  to  100  parts  of  the  solution  is  reached. 

It  is  necessary  that  the  rubbing  be  thorough  in  order  that  the  firm 
connection  between  the  bacilli  shall  be  broken  up  and  the  organisms 
distributed  throughout  the  fluid.  The  operation  usually  lasts  fifteen 
minutes.  The  fluid  is  then  placed  in  a  hand  centrifuge  and  whirled 
for  six  minutes,  then  pipeted  off,  and  rendered  feebly  alkaline  by  adding 
diluted  hydrochloric  acid  solution.  The  fluid  thus  obtained  is  too  con- 
centrated to  be  used  in  this  form,  so  must  be  diluted  with  0.5  per  cent, 
carbolic  acid  in  0.85  per  cent,  sodium  chlorid  solution.  This  solution 
should  be  repeatedly  filtered  before  receiving  the  bacillary  suspension. 
The  quantity  of  bacillary  suspension  to  be  added  should  make  the  final 
product  a  3000  dilution  of  the  original.  It  should  look  like  water  by 
transmitted  light,  but  slightly  opalescent  by  reflected  light. 

The  serum  to  be  tested  is  added  in  proportions  of  i :  10,  i :  25,  i :  50, 
i :  75,  i :  100,  i :  200,  i :  300,  etc.,  and  is  to  stand  for  twenty-four  hours. 
By  inclining  the  tube  and  looking  through  a  thin  stratum  of  the  fluid 
the  agglutinations  can  be  at  once  detected. 

Antitubercle  Serums. — Tizzoni  and  Centanni,§  Bern- 
heim,||  Paquin,**  Viqueratff  and  others  have  experimented 
in  various  ways,  hoping  that  the  principles  of  serum  therapy 
might  apply  to  tuberculosis.  Nothing  has,  however,  been 
achieved.  Maragliano's|t  antitubercle  serum  has  been  used 

*  "Deutsche  med.  Wochenschrift,"  1901,  No.  48,  p.  829. 

f  Loc.  cit. 

t  "Deutsche  med.  Wochenschrift,"  1901,  No.  48,  p.  829. 

§  "Centralbl.  f.  Bakt.,"  etc.,  Bd.  xi,  p.  82,  1892. 

||  Ibid.,  Bd.  xv,  p.  654,  1894. 
**  "New  York  Med.  Record,"  1895. 

ft  "Zur  Gewinnung  von  Antituberkulin,  Centralbl.  f.  Bakt.,"  etc., 
Nov.  5,  1896,  xx,  Nos.  1 8,  19,  p.  674. 

it  "Berliner  klin.  Wochenschrift,"  1895,  No.  32. 


Prophylaxis  747 

in  a  very  large  number  of  cases  in  human  medicine,  but  the 
glittering  results  reported  by  its  author  have  not  been  con- 
firmed. Behring*  comments  upon  it  by  saying  that  "  Ma- 
ragliano's  tubercle  antitoxin  contains  no  antitoxin." 

Babes  and  Proca,  |  Maffucci  and  di  Vestea,  {  McFarland,  § 
De  SchweinitzJI  Fisch,**  and  Patersonff  have  all  endeavored 
to  obtain  serums  of  therapeutic  value  by  immunizing  animals 
against  living  or  dead  tubercle  bacilli  or  their  products,  but 
without  success. 

From  these  discordant  observations,  the  more  favorable 
of  which  are  probably  the  hasty  records  of  inadequate  or 
incomplete  experiments,  the  conclusion  that  little  is  to  be 
hoped  from  immune  serums  in  the  treatment  of  tuberculosis 
is  inevitable. 

Prophylaxis. — It  is  the  duty  of  every  physician  to 
use  every  means  in  his  power  to  prevent  the  spread  of 
tuberculous  infection  in  the  households  under  his  care. 
To  this  end  patients  should  cease  to  kiss  the  members  of 
their  families  and  friends;  should  have  individual  knives, 
forks,  spoons,  cups,  napkins,  etc.,  carefully  kept  apart — 
secretly  if  the  patient  be  sensitive  upon  the  subject — from 
those  of  the  family,  and  scalded  after  each  meal;  should 
have  their  napkins  and  handkerchiefs,  as  well  as  whatever 
clothing  or  bed-clothing  is  soiled  by  them,  kept  apart  from 
the  common  wash,  and  boiled;  and  should  carefully  col- 
lect the  expectoration  in  a  suitable  receptacle,  that  is  ster- 
ilized or  disinfected,  without  being  permitted  to  dry,  as 
it  has  been  shown  that  the  tubercle  bacillus  can  remain 
alive  in  dried  sputum  as  long  as  nine  months.  The  phys- 
ician should  also  give  directions  for  disinfecting  the  bed- 
room occupied  by  a  consumptive  before  it  becomes  the 
chamber  of  a  healthy  person,  though  this  should  be  as 
much  the  function  of  the  municipality  as  the  disinfec- 
tion practised  after  scarlatina,  diphtheria,  and  smallpox. 

Boards  of  health  are  now  becoming  more  and  more  in- 

*  "Fortschritte  der  Med.,"  1897. 
t  "La  Med.  Moderne,"  1896,  p.  37. 
j  "Centralbl.  f.  Bakt.,"  etc.,  1896,  Bd.  xix,  p.  208. 
§  "Jour.  Amer.  Med.  Assoc.,"  Aug.  21,  1897. 

||  "Centralbl.  f.  Bakt.  und  Parasitenk.,"  Sept.  15,  1897,  Bd.  xxn, 
Nos.  8  and  9. 

**  "Jour.  Amer.  Med.  Assoc.,"  Oct.  30,  1897. 
ft  "Amer.  Medico-Surg.  Bull.,"  Jan.  25,  1898. 


748  Tuberculosis 

terested  in  tuberculosis,  and,  though  exceedingly  slow  and 
conservative  in  their  movements,  are  disseminating  litera- 
ture with  the  hope  of  achieving  by  volition  that  which  might 
otherwise  be  regarded  as  cruel  compulsion. 

So  long  as  tuberculosis  exists  among  men  or  cattle,  it 
shows  that  existing  hygienic  precautions  are  insufficient. 
While  condemning  any  unreasonable  isolation  of  patients, 
we  should  favor  the  registration  of  tuberculous  cases  as  a 
means  of  collecting  accurate  data  concerning  their  origin ;  in- 
sist upon  the  careful  domestic  sterilization  and  disinfection 
of  all  articles  used  by  the  patients;  recommend  public  disin- 
fection of  the  houses  they  cease  to  occupy;  and  approve  of 
special  hospitals  for  as  many  (especially  of  the  poorer  classes, 
among  whom  hygienic  measures  are  almost  always  opposed) 
as  can  be  persuaded  to  occupy  them. 


BOVINE  TUBERCULOSIS. 
BACILLUS  TUBERCULOSIS  Bovis. 

The  tuberculous  diseases  of  the  lower  animals  and  espe- 
cially cattle  have  lesions  closely  resembling  those  of  human 
tuberculosis,  and  containing  bacilli  similar  both  in  morphol- 
ogy and  in  staining  reaction  to  those  found  in  human  tuber- 
culosis. The  conclusion  that  they  are  identical  seems  in- 
evitable, but  in  his  monograph  upon  tuberculosis  Koch 
called  attention  to  certain  morphologic  and  cultural  differ- 
ences that  exist  between  bacilli  obtained  from  human  and 
from  animal  tuberculosis.  Unfortunately,  very  little  atten- 
tion was  paid  to  the  subject  until  Theobald  Smith*  care- 
fully compared  a  series  of  bacilli  obtained  from  human 
sputum  with  another  series  obtained  from  cattle,  horses, 
hogs,  cats,  dogs,  and  other  animals. 

His  observations  form  the  foundation  of  the  following 
description  of  the  bovine  tubercle  bacillus: 

Morphology. — The  size  of  the  bovine  bacillus  is  quite 
constant,  the  individuals  being  quite  short  (1-2  ^).  They 
are  straight,  not  very  regular  in  outline,  and  sometimes 
of  a  spindle,  sometimes  a  barrel,  and  sometimes  an  oval 
shape.  The  human  bacilli,  on  the  other  hand,  are  prone  to 
take  an  elongate  form  under  artificial  cultivation. 

*  ''Trans.  Assoc.  Amer.  Phys.,"  1896,  xi,  p.  75,  and  1898,  xm,  p.  417; 
"Jour,  of  Experimental  Medicine,"  1898,  in,  495. 


Bovine  Tuberculosis  749 

Staining. — The  bovine  bacillus  usually  stains  homoge- 
neously; the  human  bacillus  commonly  shows  the  so-called 
"  beaded  appearance." 

Vegetation. — The  human  bacillus  grows  upon  dogs'  serum 
much  more  luxuriantly  and  rapidly  than  the  bovine  bacillus. 

Metabolic  Products. — An  important  variation  in  the 
metabolism  of  the  human  and  bovine  tubercle  bacilli  has  been 
pointed  out  by  Theobald  Smith,*  who  observed  that  cultures 
of  the  two  organisms  upon  glycerin  bouillon  differed  in  the 
induced  reaction  of  the  media.  The  cultures  of  the  bovine 
bacillus  tend  toward  neutrality,  those  of  the  human  bacillus 
toward  acidity.  This  chemical  difference  is  an  adjunct  to- 
our  means  of  differentiating  the  two  organisms. 

Pathogenesis. — (a)  Guinea-pigs. — The  bovine  bacilli  are 
more  virulent  than  those  of  human  tuberculosis,  intraperi- 
toneal  inoculation  of  the  former  producing  death  in  adult 
animals  in  from  seven  to  sixteen  days;  of  the  latter,  in  from 
ten  to  thirty-eight  days.  Subcutaneous  inoculation  of  the 
bovine  bacillus  causes  death  in  less  than  fifty  days;  of  the 
human  bacillus,  in  from  fifty  to  one  hundred  days. 

(b)  Rabbits. — Rabbits  inoculated  into  the  ear  vein  with 
the  bovine  bacillus  die  in  from  seventeen  to  twenty-one  days. 
Those  receiving  human  bacilli  sometimes  live  several  months. 

(c)  Cattle. — Cows    and    heifers     receiving    intrapleural 
and  intra-abdominal  injections  of  the  human  bacilli  usually 
gain  in  weight  and  show  no  symptoms.     When  examined 
postmortem,    circumscribed     chronic    lesions     were     found. 
Those  inoculated  with  the  bovine  bacillus  lose  weight,  suffer 
from  constitutional  symptoms,  and  show  extensive  lesions 
at  the  necropsy.     Two-thirds  of  the  cattle  inoculated  experi- 
mentally with  the  bovine  bacillus  die. 

Lesions  — In  general  the  lesions  produced  by  the  bovine 
bacillus  were  rapid,  extensive,  and  necrotic.  Many  bacilli 
are  present.  Those  produced  by  the  human  bacillus  are 
more  apt  to  be  productive,  chronic,  and  contain  relatively 
few  bacilli.  The  bacilli  of  human  tuberculosis  produce  lesions 
with  many  giant  cells;  those  of  bovine  tuberculosis,  lesions 
with  rapid  coagulation  necrosis.  The  lesions  resulting  from 
the  intravenous  injection  of  human  bacilli  into  rabbits  re- 
sembled those  observed  by  Prudden  and  Hodenpyl  f  after  the 
intravenous  injection  of  boiled,  washed  tubercle  bacilli. 

*  "Trans.  Assoc.  Amer.  Phys.,"  1903,  vol.  xvm,  p.  109. 
f  "New  York  Med.  Jour.,"  June  6-20,  1891. 


750  Tuberculosis 

From  these  data  it  is  evident  that  the  bovine  bacillus 
is  by  far  the  more  virulent  and  dangerous  organism. 

At  the  International  Congress  on  Tuberculosis,  held  in 
London,  1901,  Koch  expressed  the  opinion  that  bovine 
tuberculosis  was  not  communicable  to  man.  The  matter 
is  of  the  utmost  importance  to  the  medical  profession  and 
of  far-reaching  influence  upon  many  important  sanitary 
measures  that  bear  directly  upon  the  public  health. 

Koch's  opinion,  being  opposed  to  all  that  had  been  believed 
before,  received  almost  universal  disapproval.  The  papers 
by  Arloing,*  Ravenel,|  and  Salmon  J  contain  evidence  show- 
ing that  under  certain  conditions  bovine  tuberculosis  can 
be  communicated  to  man. 

Ravenel§  has  reported  3  cases  of  accidental  cutaneous 
inoculation  of  bovine  tuberculosis  in  man.  All  were  veter- 
inary surgeons  who  became  infected  through  wounds  ac- 
cidentally inflicted  during  the  performance  of  necropsies 
upon  tuberculous  cattle.  The  tubercle  bacilli  were  demon- 
strated in  some  of  the  excised  cutaneous  nodules. 

Theobald  Smith,  ||  in  studying  3  cases  of  supposed  food 
infection,  found  what  corresponded  biologically  with  the 
human  rather  than  the  bovine  bacillus. 

In  a  later  paper  Koch**  analyzed  the  cases  usually  se- 
lected from  the  literature  to  prove  the  communicability  of 
bovine  tuberculosis  to  man,  and  showed  that  not  one  of 
the  cases  really  proves  what  is  claimed  for  it,  and  that  the 
subject  requires  further  careful  investigation  and  demon- 
stration before  it  will  be  possible  to  express  any  positive 
opinion  in  regard  to  it. 

During  the  years  that  have  elapsed  since  1901  and  the 
present  time  sentiment  has  been  almost  uniformly  against 
Koch,  and  an  enormous  literature  has  accumulated  that  in 
reality  means  very  little.  The  most  important  is  probably 
the  Royal  Commission  on  Tuberculosis  of  Great  Britain,  ft 

*"Lyon  Med.,"  Dec.  i,  1901. 

t"Univ.  of  Pa.  Bulletin,"  xiv,  p.  238,  1901;  "Lancet,"  Aug.  17 
and  19,  1901;  "Medicine,"  July  and  Aug.,  1902,  vol.  vm. 

I  "  Bull.  No.  33,  Bureau  of  Animal  Industry,"  U.  S.  Dept.  of  Agricul- 
ture, 1901. 

§  "Phila.  Med.  Jour.,"  July  21,  1900. 

||  "Amer.  Jour.  Med.  Sciences,"  Aug.  1904,  vol.  cxxvni,  No.  389, 
p.  216. 

**  Eleventh  International  Congress  for  Tuberculosis,  Berlin,  1902. 
ft  "See  the  "British  Medical  Journal,"  1907  and  1908. 


Bovine  Tuberculosis  751 

The  general  tenor  of  this  report  is  contrary  to  Koch's  views, 
and  many  believed  it  settled  the  subject.  At  the  Inter- 
national Congress  on  Tuberculosis  in  Washington,  1908, 
Koch  reviewed  the  subject  and  stated  his  continued  belief 
in  the  principle  he  had  enumerated  seven  years  before. 
Practically  the  same  contentions  were  raised  against  him 
by  much  the  same  group  of  men,  but  the  controversy  was 
more  bitter  than  before.  Koch,*  however,  leaves  us  in  no 
doubt  upon  the  subject,  summarizing  his  views  in  these 
words : 

1.  The  tubercle  bacilli  of  bovine  tuberculosis  are  different  from 

those  of  human  tuberculosis. 

2.  Human  beings  may  be  infected  by  bovine  tubercle  bacilli,  but 

serious  diseases  from  this  cause  occur  very  rarely. 

3.  Preventive    measures    against    tuberculosis    should,    therefore, 

be    directed    primarily    against    the    propagation    of    human 
tubercle  bacilli. 

He  weighed  the  contrary  evidence  that  had  been  col- 
lected during  seven  years,  showed  how  errors  had  crept 
into  the  investigations,  and  laid  down  certain  rules  to  be 
observed  before  the  experiments  could  be  accepted.  At  the 
close  of  the  congress  the  matter  remained  unsettled,  Koch 
appearing  to  have  the  best  of  the  argument. 

The  opponents  of  Koch  based  their  opinions  upon  the  sup- 
posed modifiability  of  the  tubercle  bacillus  in  different  en- 
vironments. When  it  lived  in  man,  it  was  by  virtue  of  the 
contact  with  the  human  juices  and  their  chemical  peculiarities 
compelled  to  assume  the  human  form ;  in  the  cow,  by  virtue  of 
the  different  chemical  conditions,  the  bovine  form,  etc. 
Proofs  of  this  were,  however,  wanting,  and  have  not  yet  been 
published.  On  the  other  hand,  Moriyaf  seems  to  have  shown 
that  such  changes  are  either  purely  hypothetic  or  come 
about  with  great  difficulty.  He  succeeded  in  keeping  human 
and  also  bovine  types  of  tubercle  bacilli  alive  in  tortoises 
for  twelve  months,  at  the  end  of  which  period  each  was  found 
unmodified  and  possessed  of  its  original  characteristics. 

It  was  Koch's  hope  to  be  able  to  finally  settle  the  whole 
matter,  and  to  this  end  he  asked  the  cooperation  of  many 
laboratories  throughout  different  parts  of  the  world.  Un- 
fortunately he  died  before  the  results  could  be  compiled,  but 
much  work  had  been  done  and  much  support  thereby  given 

*  "Jour.  Amer.  Med.  Assoc.,"  Oct.  10,  1908,  u,  No.  15,  p.  1256. 
t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  i.  Abt.  Orig.,  u,  1909,  460. 


752 


Tuberculosis 


his  views.  A  most  fertile  research,  the  results  of  which  form 
a  valuable  addition  to  our  knowledge  of  the  problem,  has 
been  published  by  Park  and  Krumwiede,*  who,  basing  their 
opinions  upon  the  following  tabulation  of  1224  cases, 

COMBINED  TABULATION  CASES  REPORTED  AND  OWN  SERIES  OF  CASES. 


Diagnosis. 

Adults  1  6  years 
and  over. 

Children  5  to  16 
years. 

Children  under 
5  years. 

H. 

B. 

H. 

B. 

H. 

B. 

Pulmonary  tuberculosis  
Tuberculous  adenitis,  axillary 
or  inguinal  

644 

2 
27 
14 

6 
29 

5 

27 
17 
3 

_! 

(I?) 

I 

4 

i 

i 

i 

1  1 

4 
36 

8 

2 

4 

i 

7 
3 

38 

2 

I 

21 
7 

3 
i 

3 
i 

23 

2 
15 
9 

13 

43 

3 

52 
27 

26 

21 
13 

12 

5 

8 

i 

4 

Tuberculous  adenitis,  cervical 
Abdominal  tuberculosis  
Generalized  tuberculosis,  ali- 
mentary origin.  

Generalized  tuberculosis  
Generalized    tuberculosis    in- 
cluding meninges,  aliment- 
ary origin  

Generalized    tuberculosis    in- 
cluding meninges  
Tubercular  meningitis  
Tuberculosis    of    bones    and 
joints  

Genito-urinary  tuberculosis.  . 
Tuberculosis  of  skin  
Miscellaneous  cases: 
Tuberculosis  of  tonsils  .... 
Tuberculosis  of  mouth  and 
cervical  nodes  
Tuberculous  sinusor  abscess 
Sepsis,  latent  bacilli  

Totals  .  . 

777 

10 

117 

36 

2I.S 

65 

Mixed  or  double  infections,  4  cases. 

Total  cases,  1224. 

come  to  the  following  conclusions : — 

Conclusions. — Bovine  tuberculosis  is  practically  a  negligi- 
ble factor  in  adults.  It  very  rarely  causes  pulmonary  tuber- 
culosis or  phthisis  which  causes  the  vast  majority  of  deaths 
from  tuberculosis  in  man,  and  is  the  type  of  disease  respon- 
sible for  the  spread  of  the  virus  from  man  to  man. 

In  children,  however,  the  bovine  type  of  tubercle  bacillus 
causes  a  marked  percentage  of  the  cases  of  cervical  adenitis, 
leading  to  operation,  temporary  disablement,  discomfort,  and 

*  "Journal  of  Medical  Research,"  1910,  xxin,  No.  2,  p.  205;  191 1, 
xxv,  No.  2,  p.  313. 


Bovine  Tuberculosis  753 

disfigurement.  It  causes  a  large  percentage  of  the  rarer 
types  of  alimentary  tuberculosis  requiring  operative  inter- 
ference or  causing  the  death  of  the  child  directly  or  as  a 
contributing  cause  in  other  diseases. 

In  young  children  it  becomes  a  menace  to  life  and  causes 
from  6  J  to  10  per  cent,  of  the  total  fatalities  from  this  disease. 

Prophylaxis. — The  prevention  of  tuberculosis  in  cattle  is 
a  matter  of  vast  sanitary  importance.  Not  only  have  we 
to  consider  the  danger  of  infection  from  milk  containing 
tubercle  bacilli,  but  also  the  inferior  quality  and  diminished 
usefulness  of  milk  and  flesh  coming  from  animals  that  are 
diseased.  The  extermination  of  bovine  tuberculosis,  there- 
fore, becomes  imperative,  and  the  utmost  efforts  should  be 
made  to  bring  it  about.  Several  separate  measures  must 
be  considered: 

1.  Improvement  in  the  methods  of  diagnosis,  by  which 
the  recognition  of  the  disease  is  made  possible  before  its 
ravages  are  great.     This  is  rapidly  coming  about  with  in- 
creasing information  regarding  the  use  and  abuse  of  tu- 
berculin, etc. 

2.  Means  by  which  infected  animals  shall  be  destroyed. 
Here  the  municipal  and  state  governments  furnish  inade- 
quate funds  to  make  possible  the  destruction  of   diseased 
cattle  without  adequate  compensation — an  injustice  to  the 
unfortunate  owner. 

3.  Means   of   preventing   the   infection   of  healthy   ani- 
mals.    In  many  places  this  is  being  achieved  with  brilliant 
success  by  separation  of  the  herd,  healthy  and  newly  born 
animals  constituting  one  part,  suspicious  animals  the  other. 
By  these  means  valuable  breeding  animals  can  be  kept  for 
a  time,  at  least,  in  usefulness.     A  second  and  less  successful 
means  of  preventing  infection  is  by  means  of  prophylactic 
vaccination   of   the   healthy    animals   with    dead   cultures, 
modified  living  cultures,  or  by  bacteriotoxins  made  by  com- 
minuting them. 

Experiments  of  this  kind  have  been  conducted  by  Mc- 
Fadyen,*  on  a  large  scale  by  von  Behring,  f  by  Pearson 
and  Gilliland,  t  Calmette  and  Guerin,§  and  by  Theobald 

c  "Jour.  Comp.  Path,  and  Therap.,"  June,  1901. 

f  "Beitrage  zur  experimentellen  Therapie,"  1902,  Hft.  5. 

|  "Jour,  of  Comp.  Med.  Vet.  Archiv,"  Nov.  1902,  "Univ.  of  Penna. 
Med.  Bull.,"  April,  1905. 

§  "Ann.  de  1'Inst.  Pasteur.,"  Oct.,  1905,  May,  1906,  and  July,  1907; 
and  "International  Congress  on  Tuberculosis,"  Washington,   1908. 
48 


754  Tuberculosis 

Smith,*  all  of  whom  think  distinct  resisting  power  against 
infection  by  the  tubercle  bacillus  can  thus  be  brought  about. 

Tuberculin  Test  for  Tuberculosis  of  Cattle. — The  febrile 
reaction  caused  by  the  injection  of  tuberculin  into  tubercu- 
lous animals  is  an  important  adjunct  to  our  means  of  diag- 
nosticating the  disease.  For  the  recognition  of  tuberculosis 
in  cattle  it  is  easily  carried  out. 

To  make  a  satisfactory  diagnostic  test  the  temperature  of 
the  animal  should  be  taken  every  few  hours  for  a  day  or  two 
before  the  tuberculin  is  administered,  in  order  that  the  nor- 
mal diurnal  and  nocturnal  variations  of  temperature  shall 
be  known.  The  tuberculin  is  then  administered  by  hypoder- 
mic injection  into  the  shoulder  or  flank,  and  the  tem- 
perature subsequently  taken  every  two  hours  for  the  next 
twenty-four  hours.  A  reaction  of  two  degrees  beyond  that 
normal  to  the  individual  animal  is  positive  of  tuberculosis. 
After  one  reaction  of  this  kind  the  animal  will  not  again 
react  to  an  equal  dose  of  tuberculin  for  a  number  of  weeks. 


FOWL  TUBERCULOSIS. 
BACILLUS  TUBERCULOSIS  AVIUM. 

The  occasional  spontaneous  occurrence  of  tuberculosis  in 
chickens,  parrots,  ducks,  and  other  birds,  observed  as  early 
as  1868  by  Rolofff  and  Paulicki,  {  was  originally  attributed 
to  Bacillus  tuberculosis  hominis,  but  the  work  of  Rivolta,§ 
Mafucci,||  Cadio,  Gilbert  and  Roger,**  and  others  has  shown 
that,  while  similar  to  it  in  many  respects,  the  organism 
found  in  the  avian  diseases  has  distinct  peculiarities  which 
make  it  a  different  variety,  if  not  a  separate  species.  Cadio, 
Gilbert,  and  Roger  succeeded  in  infecting  fowls  by  feeding 
them  upon  food  containing  tubercle  bacilli,  and  keeping 
them  in  cages  in  which  dust  containing  tubercle  bacilli  was 
placed.  The  infection  was  aided  by  lowering  the  tempera- 
ture of  the  birds  with  antipyrin  and  lessening  their  vitality 
by  starvation. 

*  ''Journal  of  Medical  Research,"  June,  1908,  xvm,  No.  3,  p.  451. 
f'Mag.  f.  d.  ges.  Tierheilkunde,"  1868. 
t  "Beitr.  zur  vergl.  Anat.,"  Berlin,  1872. 
§  "Giorn.  anat.  fisiol.  e.  path.,"  Pisa,  1883. 
||  "Zeitschrift  fur  Hygiene,"  Bd.  xi. 
**  "La  Semaine  medicale,"  1890,  p.  45. 


Fowl  Tuberculosis  755 

Morphologic  Peculiarities. — Morphologically,  the  organ- 
ism found  in  avian  tuberculosis  is  similar  to  that  found 
in  the  mammalian  disease,  but  is  a  little  longer  and  more 
slender,  with  more  marked  tendency  to  club  and  branched 
forms.  Fragmented  and  beaded  forms  occur  as  in  the 
human  tubercle  bacilli. 

Staining. — The  avian  bacillus  stains  in  about  the  same 
manner  as  the  human  and  bovine  bacilli  and  has  an  equal 
resistance  to  the  decolorant  effect  of  acids. 

Cultivation. — Marked  rapidity  and  luxuriance  of  growth 
are  characteristic  of  the  avian  bacillus,  which  grows  upon 
agar-agar  and  ordinary  bouillon  prepared  without  glycerin. 


Fig.  247. — Bacillus  tuberculosis  avium. 

The  growth  also  lacks  the  dry  quality  characteristic  of 
cultures  of  the  human  and  bovine  bacilli.  Old  cultures  of 
the  bacillus  of  fowl  tuberculosis  turn  slightly  yellow. 

Thermic  Sensitivity. — The  bacillus  also  differs  in  its 
thermic  sensitivity  and  will  grow  at  42°  to  45°  C.  quite  as 
well  as  at  37°  C.,  while  the  growth  of  the  human  and  mam- 
malian bacilli  ceases  at  42°  C.  Moreover,  growth  at  43°  C. 
does  not  attenuate  its  virulence.  The  thermal  death- 
point  is  70°  C.  Upon  culture-media  it  is  said  to  retain  its 
virulence  as  long  as  two  years. 

Pathogenesis. — Birds  are  the  most  susceptible  animals 
for  experimental  inoculation,  the  embryos  and  young  being 


756  Tuberculosis 

more  susceptible  than  the  adults.  Artificial  inoculation 
can  be  made  in  the  subcutaneous  tissue,  in  the  trachea,  and 
in  the  veins;  never  through  the  intestine.  After  inoculation 
the  birds  die  in  from  one  to  seven  months.  The  chief  seat 
of  the  disease  is  the  liver,  where  cellular  (lymphocytic)  nodes, 
lacking  the  central  coagulation  and  the  giant-cell  formation 
of  mammalian  tuberculosis,  and  enormously  rich  in  bacilli, 
are  found.  The  disease  never  begins  in  the  lungs,  and  the 
fowls  that  are  diseased  never  show  bacilli  in  the  sputum  or 
in  the  dung. 

Guinea-pigs  are  quite  immune,  or  after  inoculation  develop 
cheesy  nodes,  but  do  not  die. 

Rabbits  are  easily  infected,  an  abscess  forming  at  the 
seat  of  inoculation,  nodules  forming  later  in  the  lungs,  so 
that  the  distribution  is  quite  different  from  that  seen  in 
birds.  It  is  possible  that  the  avian  bacillus  occasionally 
infects  man. 

The  possibility  that  this  bacillus  is  derived  from  the  same 
stock  as  the  tubercle  bacillus  is  strengthened  by  the  experi- 
ments of  Fermi  and  Salsano,*  who  succeeded  in  increasing 
its  virulence  until  it  became  fatal  to  guinea-pigs  by  adding 
glucose  and  lactic  acid  to  the  cultures  inoculated. 

FISH  TUBERCULOSIS. 

Dubarre  and  Terref  isolated  a  bacillus  having  the  tinctorial 
and  morphologic  characteristics  of  the  tubercle  bacillus 
from  carp  suffering  from  a  tubercle-like  affection.  In  respect 
to  cultivation,  however,  it  was  unlike  tubercle  bacilli,  growing 
readily  upon  simple  culture-media  at  15°  to  30°  C.,  and  not 
at  37°  C. 

Weber  and  TaubeJ  found  the  same  organism,  or  what 
seemed  to  be  the  same  organism,  in  mud  and  in  a  healthy  frog. 

BACILLI  RESEMBLING  THE  TUBERCLE  BACILLUS. 

It  is  not  improbable  that  the  bacilli  of  human,  bovine, 
and  avian  tuberculosis  are  closely  related  to  one  another,  and, 
together  with  a  few  other  micro-organisms  of  similar  mor- 
phology and  staining  peculiarities,  have  a  common  ancestry 

""Centralbl.  f.  Bakt.,"  etc.,  xn,  750. 
t  "Compt.  rendu  de  la  Soc.  de  Biol.  de  Pari,"  1897,  446. 
I  "Tuberkulose  Arbeiten  aus  dem  Kaiserlichen  Gesundheitsamte," 
1905. 


Bacillus  Smegmatis  757 

and  are  descended  from  the  same  original  stock.  The 
most  important  of  these  similar  organisms  are  Bacillus 
leprce  (q.  v.),  B.  smegmatis,  and  Moeller's  grass  bacillus. 

BACILLUS  SMEGMATIS. 

Alvarez  and  Tavel,*  Matterstock,  f  Klemperer  and  Bittu,  J 
Cowie,  §  and  others  have  described  peculiar  bacilli  in  smegma 
taken  from  the  genitals  of  man  and  the  lower  animals,  as  well 
as  from  the  moist  skin  in  the  folds  of  the  groin,  the  axillae, 
and  the  anus.  They  are  also  sometimes  found  in  urine,  and 
occasionally  in  the  saliva  and  sputum. 

Morphology  and  Staining. — The  organisms  are  of  some- 
what variable  morphology,  but  in  general  resemble  the  tuber- 
cle bacillus,  stain  with  carbol-fuchsin,  as  does  the  tubercle 
bacillus,  and  resist  the  decolorant  action  of  acids.  They  are, 
however,  decolorized  by  absolute  alcohol,  though  Moeller  de- 
clares the  smegma  bacillus  to  be  absolutely  alcohol-proof  as 
well  as  acid-proof,  and  admits  no  tinctorial  difference  between 
it  and  the  tubercle  bacillus.  The  bacillus,  being  about  the 
size  and  shape  of  the  tubercle  bacillus,  is  very  readily  mis- 
taken for  it,  and  its  presence  in  cases  of  suspected  tubercu- 
losis of  the  genito-urinary  apparatus,  and  in  urine  and  other 
secretions  in  which  it  is  likely  to  be  present,  may  lead  to 
considerable  confusion.  The  final  differentiation  may  have 
to  rest  upon  animal  inoculation. 

Cultivation. — The  cultivation  of  the  smegma  bacillus  is 
difficult  and  was  first  achieved  by  Czaplewski.||  Doutrele- 
pont  and  Matterstock  cultivated  it  upon  coagulated  hydro- 
cele  fluid,  but  were  unable  to  transplant  the  growth  suc- 
cessfully. 

Novy**  recommends  the  cultivation  of  the  smegma 
bacillus  by  inoculating  a  tube  of  melted  agar-agar  cooled 
to  50°  C.  with  the  appropriate  material,  and  mixing  with 
it  about  2  c.c.  of  blood  withdrawn  from  a  vein  of  the  arm 
with  a  sterile  hypodermic  syringe.  The  blood-agar  mixture 
is  poured  into  a  sterile  Petri  dish  and  set  aside  for  a  day 

*"Archiv  de  Physiol.  norm,  et  Path.,"  1885,  No.  7. 
f  "Mittheil.  aus  d.  med.  Klin.  d.  Univ.  d.  Wiirzburg,"  1885,  Bd.  VI. 
J  "Virchow's  Archives,"  v,  103. 

§  "Journal  of  Experimental  Medicine,"  vol.  v,  1900-01,  p.  205. 
||  "Miinchener  med.  Wochenschrift,"  1897. 
**  " Laboratory  Work  in  Bacteriology,"  1899. 


758  Tuberculosis 

or  two  at  37°  C.  The  colonies  that  form  are  to  be  examined 
for  bacilli  that  resist  decolorization  with  acids. 

Moeller*  found  it  comparatively  easy  to  secure  cultures 
of  the  smegma  bacillus  by  a  peculiar  method.  To  secure 
small  quantities  of  human  serum  for  the  purpose  of  investi- 
gating the  phenomena  of  agglutination  he  applied  small 
cantharidal  blisters  to  the  skins  of  various  healthy  and 
other  men,  and  found  large  numbers  of  acid-proof  bacilli  in 
the  serum  saturated  with  epithelial  substance,  that  re- 
mained after  most  of  the  serum  had  been  withdrawn.  He 
removed  the  skin  covering  from  the  blister,  placed  it  in  the 
remaining  serum,  and  kept  it  in  the  incubator  for  three  or 
four  days,  after  which  he  found  a  dry,  floating  scum,  which 
consisted  of  enormous  numbers  of  the  bacilli,  upon  the  serum. 
From  this  growth  he  was  subsequently  able  to  start  cultures 
of  the  smegma  bacillus  upon  glycerin  agar-agar.  Human 
blood-serum  is  thus  found  to  be  the  best  medium  upon 
which  to  start  the  culture. 

Agar. — A  culture  thus  isolated  grew  upon  all  the  usual 
culture-media.  Upon  glycerin-agar,  at  37°  C.,  the  colonies 
appeared  as  minute,  dull,  grayish- white,  dry,  rounded  scales, 
which  later  became  lobulated  and  velvety.  At  room  tem- 
perature the  dry  appearance  of  the  growth  was  retained. 
The  water  of  condensation  remains  clear. 

Potato. — On  potato  the  growth  was  luxuriant,  grayish, 
and  dull. 

Milk. — Milk  is  said  to  be  an  exceptionally  good  medium, 
growth  taking  place  in  it  with  rapidity  and  without  coagu- 
lation. 

Bouillon. — The  growth  forms  a  dry  white  scum  upon  the 
surface,  the  medium  remaining  clear. 

Pathogenesis. — So  far  as  is  known,  the  smegma  bacillus 
is  a  harmless  saprophyte. 

MOELLER'S  GRASS  BACILLUS. 

Bacilli  found  in  milk,  butter,  timothy  hay,  cow-dung,  etc., 
which  stain  like  the  tubercle  bacillus  and  may  be  mistaken 
for  it,  have  been  described  by  Moeller. f  The  organisms  so 
closely  resemble  the  tubercle  bacillus  that  guinea-pig  inocu- 

*"Centralbl.  f.  Bakt.  u.  Parasitenk"  (Originale),  Bd.  xxxi,  No.  7, 
p.  278,  March  12,  1902. 

t  "Deutsche  med.  Zeitung,"  1898,  p.  135;  "Deutsche  med.  Wochen- 
schrift,"  1898,  p.  376,  etc. 


Bacilli  Resembling  the  Tubercle  Bacillus       759 

lations  must  be  resorted  to  in  cases  of  doubt,  but  as  some  of 
these  organisms  sometimes  kill  the  guinea-pigs  after  a 
month  or  two,  and  as  small  nodules  or  tubercles  may  be 
present  in  the  mesentery,  peritoneum,  liver,  lung,  etc.,  of 
such  animals,  the  diagnosis  may  have  to  be  subjected  to  the 
further  confirmation  of  a  histologic  examination  of  the 
lesions  in  order  to  exclude  tuberculosis.  In  cases  of  this 
kind  it  should  not  be  forgotten  that  the  tubercle  bacillus 
can  be  present  in  the  substances  mentioned,  so  that  the 
exact  differentiation  becomes  a  very  fine  one.  An  instruc- 
tive study  of  these  organisms  has  been  made  by  Abbott 
and  Gildersleeve,*  who,  in  an  elaborate  work  upon  the 
"  Etiological  Significance  of  the  Acid-resisting  Group  of 
Bacteria,  and  the  Evidence  in  Favor  of  their  Botanical 
Relation  to  Bacillus  Tuberculosis,"  a  work  that  gives  com- 
plete references  to  the  literature  of  the  subject,  come  to  the 
following  conclusions : 

1.  That   the  majority  of  the  acid-resisting  bacteria  may  be  dis- 
tinguished from  true  tubercle  bacilli  by  their  inability  to  resist  decolor- 
ization  by  a  30  per  cent,  solution  of  nitric  acid  in  water. 

2.  That  some  of  the  acid-resisting  bacteria  are  capable  of  causing 
in  rabbits   and   guinea-pigs    nodular  lesions  suggestive   of  tubercles; 
that  these  lesions,  while  often  very  much  like  tubercles  in  their  histo- 
logic structure,  may  nevertheless  usually  be  distinguished  from  them  by 
the  following  peculiarities: 

(a)  When  occurring  as  a  result  of  intravenous  inoculation,  they  are 
always  seen  in  the  kidneys,  only  occasionally  in  the  lungs,  and  practi- 
cally not  at  all  in  the  other  organs. 

(b)  They  constitute  a  localized  lesion,  having  no  tendency  to  dis- 
semination, metastasis,  or  progressive  destruction  of  tissue  by  casea- 
tion. 

(c}  They  tend  to  terminate  in  suppuration  or  organization  rather  than 
in  progressive  caseation,  as  is  the  case  with  true  tubercles. 

(d)  They  are  more  commonly  and  conspicuously  marked  by  the  ac- 
tinomyces  type  of  development  of  the  organisms  than  is  the  case  with 
true  tubercles,  and  these  actinomycetes  are  less  resistant  to  decoloriza- 
tion  by  strong  acid  solutions  than  are  those  occasionally  seen  in  tubercles. 

3.  That  by  subcutaneous,  intravenous,  and  intrapulmonary  inocula- 
tion of  hogs  (4)  and  calves  (15)  the  typical  members  of  the  acid-resisting 
group  are  incapable  of  causing  lesions  in  any  way  suggestive  of  those  re- 
sulting from  similar  inoculations  of  the  same  animals  with  true  tubercle 
bacilli. 

4.  That  though  occasionally  present  in  dairy  products,  they  are  to  be 
regarded  as  of  no  significance,  etiologically  speaking,  but  may  be  con- 
sidered as  accidental  contaminations  from  the  surroundings,  and  not  as 
evidence  of  disease  in  the  animals. 

5.  That  the  designation  "bacillus"  as  applied  to  this  group  of  bac- 
teria and  to  the  exciter  of  tuberculosis  is  a  misnomer;  they  are  more  cor- 
rectly classified  as  actinomyces. 

*"Univ.  of  Penna.  Bulletin,"  June,  1902. 


760  Tuberculosis 


THE  BUTTER  BACILLUS. 

Petri,*  Rabinovitsch,  |  and  KornJ  have  described,  as 
Bacillus  butyricus,  an  acid-fast  organism  morphologically 
like  the  tubercle  bacillus,  which  may  at  times  be  found  in 
butter.  Its  chief  importance  lies  in  the  confusion  that  may 
arise  through  mistaking  it  for  the  tubercle  bacillus  where 
attention  is  paid  to  the  morphologic  and  tinctorial  characters 
only,  as  tubercle  bacilli  may  be  found  in  butter  made  from 
cream  from  the  milk  of  tuberculous  cattle. 

Isolation  and  cultivation  of  these  organisms  is  easy,  and 
more  than  any  other  measure  serves  to  differentiate  them 
from  the  tubercle  bacillus,  as  they  grow  upon  nearly  all  the 
culture-media  with  rapidity  and  luxuriance. 

PSEUDOTUBERCULOSIS. 
BACILLUS    PSEUDOTUBERCULOSIS. 

Pfeiffer,§  Malassez  and  Vignal,||  Eberth,**  Chantemesse,tt 
Charrin,  and  Roger  it  have  all  reported  cases  of  so-called 
pseudotuberculosis  occurring  in  guinea-pigs,  and  character- 
ized by  the  formation  of  cellular  nodules  in  the  liver  and 
kidneys  much  resembling  miliary  tubercles.  Cultures  made 
from  them  showed  the  presence  of  a  small  motile  bacillus 
which  could  easily  be  stained  by  ordinary  methods.  When 
introduced  subcutaneously  into  guinea-pigs,  the  original  dis- 
ease was  reproduced. 

Morphology  and  Cultivation. — Bacillus  pseudotuber- 
culosis is  characterized  by  Pfeiffer  as  follows :  The  organism  is 
rod-shaped,  the  rods  varying  in  length  (0.4  to  1.2  {i}  and 
sometimes  united  in  chains.  They  may  be  almost  round, 
and  then  resemble  diplococci.  They  stain  by  ordinary 
methods,  but  not  by  Gram's  method.  They  are  motile  and 
have  flagella  like  the  typhoid  and  colon  bacilli.  They  form 

*  "Arbeiten  aus  dem  Kaiselichen  Gesundheitsamt,"  1897. 

t  "Zeitschrift  fur  Hygiene,"  etc.,  1897. 

t  "Centralbl.  f.  Bakt."  etc.,  1899. 

\  "Bacillare  tuberculose,  u.  s.  w.,"  Leipzig,  1889. 

j|  "Archiv.  de  Physiol.  norm,  et  Path.,"  1883  and  1884. 
**  "Virchow's  Archiv,"  Bd.  en. 
ft  ''Ann.  de  1'Inst.  Pasteur,"  1887. 
tJ  "Compte-rendu  de  1'Acad.  des  Sci.,"  Paris,  t.  cvi. 


Pseudotuberculosis  761 

no  spores.  Upon  gelatin  and  agar-agar  circular  colonies 
with  a  dark  nucleus  surrounded  by  a  transparent  zone  are 
formed.  In  gelatin  punctures  the  bacilli  grow  all  along  the 
line  of  puncture  and  form  a  surface  growth  with  concentric 
markings.  The  gelatin  is  not  liquefied.  The  bacilli  grow 
readily  upon  agar  and  on  potato,  but  without  character- 


Fig.    248. — Bacillus    pseudotuberculosis    from    agar-agar.       X     1000 
(Itzerott  and  Niemann). 

istic  appearances.     In  bouillon  a  diffuse  turbidity  occurs, 
with  floating  and  suspended  flakes.     Milk  is  not  altered. 

Pathogenesis. — The  bacillus  is  fatal  to  mice,  guinea- 
pigs,  rabbits,  hares,  and  other  rodents  in  about  twenty 
days  after  inoculation.  At  the  seat  of  inoculation  an  abscess 
develops,  the  neighboring  lymphatic  glands  enlarge  and 
caseate,  and  nodules  resembling  tubercles  form  in  the 
internal  organs.  Similar  bacilli  studied  by  Pfeiffer  were 
isolated  from  a  horse  supposed  to  have  glanders. 


CHAPTER   XXVIII. 
LEPROSY. 


BACILLUS  LEPR^  (HANSEN).* 

General  Characteristics.  —  A  non-motile,  non-flagellate,  non-spor- 
ogenous,  chromogenic,  non-liquefying,  non-aerogenic,  distinctly  aerobic, 
parasitic  and  highly  pathogenic,  acid-resisting  bacillus,  staining  by 
Gram's  method,  and  cultivable  upon  specially  prepared  artificial  media. 
It  does  not  form  indol,  or  acidulate  or  coagulate  milk. 

Leprosy  very  early  received  attention  and  study.  Moses 
included  in  the  laws  to  the  people  of  Israel  rules  for  its  diag- 
nosis, for  the  isolation  of  the  sufferers,  for  the  determination 
of  recovery,  and  for  the  sacrificial  observances  to  be  fulfilled 
before  the  convalescent  could  once  more  mingle  with  his  peo- 
ple. The  Bible  is  replete  with  miracles  wrought  upon  lepers, 
and  during  the  times  of  biblical  tradition  it  seems  to  have 
been  an  exceedingly  common  and  malignant  disease.  Many 
of  the  diseases  called  leprosy  in  the  Bible  were,  however,  in 
all  probability,  less  important  parasitic  skin  affections. 

Distribution.  —  At  the  present  time,  although  we  hear 
very  little  about  it  in  the  northern  United  States,  leprosy  is 
a  widespread  disease  and  exists  much  the  same  as  it  did 
several  thousand  years  ago  in  Palestine,  Syria,  Egypt, 
and  the  adjacent  countries,  and  is  common  in  China,  Japan, 
and  India.  South  Africa  has  many  cases,  and  Europe,  es- 
pecially Norway,  Sweden,  and  parts  of  the  Mediterranean 
coast,  a  considerable  number.  In  certain  islands,  especially 
the  Sandwich  and  Philippine  Islands,  it  is  endemic.  In 
the  United  States  the  disease  is  uncommon,  the  Southern 
States  and  Gulf  coast  being  chiefly  affected. 

A  commission  of  the  Marine-  Hospital  Service,  formed  for 
the  purpose  of  investigating  the  prevalence  of  leprosy,  in 
1902  reported  278  existing  cases  in  the  United  States.  Of 
these,  155  occurred  in  the  State  of  Louisiana.  The  other 
States  with  numerous  cases  were  California,  24;  Florida,  24; 
Minnesota,  20;  and  North  Dakota,  16.  No  other  State 
had  more  than  7  (New  York).  Of  the  cases,  145  were 
American  born,  120  foreign  born,  the  remainder  uncertain 

*  "Virchow's  Archives,"  1879. 
762 


Staining 


763 


Etiology. — The  cause  of  leprosy  is,  without  doubt,  the 
lepra  bacillus,  discovered  by  Hansen  in  1879. 

Morphology. — The  bacillus  is  about  the  same  size  as 
the  tubercle  bacillus.  Its  protoplasm  commonly  presents 
open  spaces  of  fractures,  giving  it  a  beaded  appearance,  like 
the  tubercle  bacillus.  It  occurs  singly  or  in  irregular  groups. 
There  is  no  characteristic  grouping  and  filaments  are  un- 
known. It  is  not  motile  and  has  no  flagella  and  no  spores. 

Duval  found  that  the  cultivated  bacilli  are  longer,  more 
curved,  and  show  a  greater  irregularity  in  the  distribution  of 
the  chromatin  than  those  in  the  tissues  where  they  are  short, 
slender,  and  slightly  curved.  In  artificial  cultures  there  is  a 


Fig.  249. — Lepra  bacilli.     Smear  from  a  lepra  node  stained  with  carbol- 
fuchsin  (Kolle  and  Wassermann). 


delicate  filamentous  arrangement  of  the  bacilli,  especially 
where  they  have  become  accustomed  to  a  saprophytic  exist- 
ence. They  often  contain  distinct  metachromatic  granules 
analogous  to  those  met  with  in  certain  forms  of  the  diphtheria 
bacillus.  They  are  quite  pleomorphous,  and  in  the  same  cul- 
ture all  forms  occur,  from  solidly  staining  coccoid  shapes  to 
slender  slightly  curved  filaments,  with  numerous  chromatic 
segments  and  occasionally  metachromatic  granules.  Some- 
times the  organisms  are  pointed  at  the  ends. 

Staining. — It  stains  in  very  much  the  same  way  as  the 
tubercle  bacillus,  but  permits  of  a  more  ready  penetra- 
tion of  the  stain,  so  that  the  ordinary  aqueous  solutions 


764 


Leprosy 


of  the  anilin  dyes  color  it  quite  readily.  The  property  of 
retaining  the  color  in  the  presence  of  the  mineral  acids  also 
characterizes  the  lepra  bacillus,  and  the  methods  of  Ehrlich, 
Gabbet,  and  Unna  for  staining  the  tubercle  bacillus,  can 
be  used  for  its  detection.  It  stains  well  by  Gram's  method 
and  by  Weigert's  modification  of  it,  by  which  beautiful 
tissue  specimens  can  be  prepared. 


Fig.  250. — Section  of  one  of  the  nodules  from  the  patient  shown  in 
Fig.  252,  stained  by  the  Weigert-Gram  method  to  show  the  lepra  ba- 
cilli scattered  through  the  tissue  and  inclosed  in  the  large  vacuolated 
"lepra-cells."  Magnified  1000  diameters. 


Czaplewski  found  that  the  lepra  bacilli  in  his  cultures 
colored  uniformly  when  young,  but  were  invariably  granular 
when  old.  The  more  rapidly  the  organism  grew,  the  more 
slender  it  appeared. 


Cultivation  765 

Cultivation. — Many  endeavors  have  been  made  to  culti- 
vate this  bacillus  upon  artificially  prepared  media,  but  in 
1903  Hansen,*  who  discovered  the  organism,  declares  that 
no  one  had  yet  cultivated  it. 

Bordoni-Uffredozzif  was  able  to  cultivate  a  bacillus  which 
partook  of  the  staining  peculiarities  of  the  lepra  bacillus  as 
it  appears  in  the  tissues,  but  differed  in  morphology. 

CzaplewskiJ  confirmed  the  work  of  Bordoni-Uffredozzi, 
and  described  a  bacillus  supposed  to  be  the  lepra  bacillus, 
which  he  'succeeded  in  cultivating  from  the  nasal  secretions 
of  a  leper. 

The  bacillus  was  isolated  upon  a  culture-medium  consist- 
ing of  glycerinized  serum  without  the  addition  of  salt, 
peptone,  or  sugar.  The  mixture  was  poured  into  Petri 
dishes,  coagulated  by  heat,  and  sterilized  by  the  inter- 
mittent method. 

The  secretion,  being  rich  in  lepra  bacilli,  was  taken  up  with 
a  platinum  wire  and  inoculated  upon  the  culture-medium 
by  a  series  of  linear  strokes.  The  dishes  were  then  sealed 
with  paraffin  and  kept  in  the  incubating  oven  at  37°  C. 

Numerous  colonies,  chiefly  of  Staphylococcus  pyogenes 
aureus  and  the  bacillus  of  Friedlander,  developed,  and  in 
addition  a  number  of  strange  colonies,  composed  of  slender 
bacilli  about  the  size  and  form  of  the  lepra  bacillus. 

These  colonies  were  grayish  yellow,  humped  in  the  middle, 
i  to  2  mm.  in  diameter,  irregularly  rounded,  and  uneven  at 
the  edges.  They  were  firm  and  could  be  entirely  inverted 
with  the  platinum  wire,  although  the  consistence  was 
crumbly.  They  were  excavated  on  the  under  side. 

The  colonies  that  form  upon  agar-agar  are  much  like  those 
described  by  Bordoni-Uffredozzi,  and  appear  as  isolated, 
grayish,  rounded  flakes,  thicker  in  the  center  than  at  the 
edges,  and  characterized  by  an  irregular  serrated  border 
from  which  a  fine  irregular  network  extends  upon  the 
medium.  These  projections  consist  of  bundles  of  the  bacilli. 

When  a  transfer  was  made  from  one  of  these  colonies 
to  fresh  media,  the  growth  became  apparent  in  a  few  days 

*  Kolle  and  Wassermann's  "Handbuch  der  pathogenen  Mikroorgan- 
ismen,"  n,  p.  184,  1903. 

t  "Zeitschrift  f.  Hygiene,"  etc.,   1884,  in. 

t  "Centralbl.  f.  Bakt.  und  Parasitenk.,"  Jan.  31,  1898,  vol.  xxm,  Nos. 
3  and  4,  p.  97. 


766  Leprosy 

and  assumed  a  band-like  form,  with  a  plateau-like  elevation 
in  the  center. 

The  bacillus  thus  isolated  grew  with  moderate  rapidity 
upon  all  the  ordinary  culture-media  except  potato.  Upon 
blood-serum  the  growth  was  more  luxuriant  and  fluid 
than  upon  the  solid  media.  Upon  coagulated  serum  the 
growth  was  somewhat  dry  and  elevated,  and  was  frequently 
so  loosely  attached  to  the  surface  of  the  medium  as  to 
be  readily  lifted  up  by  the  platinum  wire. 

The  growth  was  especially  luxuriant  upon  sheep's  blood- 
serum  to  which  5  per  cent,  of  glycerin  was  added.  The 
growth  upon  the  Loffler  mixture  was  also  luxuriant. 

Upon  agar-agar  the  growth  is  more  meager;  it  is  more 
luxuriant  upon  glycerin  agar-agar  than  upon  plain  agar- 
agar,  the  bacterial  mass  appearing  grayish  and  flatter  than 
upon  blood-serum.  The  growth  never  extends  to  the 
water  of  condensation  to  form  a  floating  layer. 

The  bacillus  develops  well  upon  gelatin  after  it  has  grown 
artificially  for  a  number  of  generations  and  become  accus- 
tomed to  a  saprophytic  existence.  Upon  the  surface  of  gela- 
tin the  growth  is,  in  general,  similar  to  that  upon  agar-agar. 
In  puncture  cultures  most  of  the  growth  occurs  upon  the 
surface  to  form  a  whitish,  grayish,  or  yellowish  wrinkled 
layer.  Below  the  surface  of  the  gelatin  the  growth  occurs  as 
a  thick,  granular  column.  The  medium  is  not  liquefied. 

In  bouillon,  growth  occurs  only  at  the  bottom  of  the  tube 
in  the  form  of  a  powdery  sediment. 

Spronck*  believed  that  he  had  successfully  cultivated  the 
organism  upon  glycerinized,  neutralized  potatoes,  first  seeing 
the  growth  after  the  lapse  of  ten  days.  Cultures  thus  pre- 
pared were  found  to  be  agglutinated  by  the  blood-serum  of 
lepra  cases,  and  he  recommends  the  agglutination  test  for 
the  diagnosis  of  obscure  cases  of  the  disease. 

Ducrey  seems  to  have  cultivated  the  lepra  bacillus  in 
grape-sugar,  agar,  and  in  bouillon  in  vacuo.  His  results 
need  confirmation. 

Rostf  claimed  to  isolate  and  cultivate  the  lepra  bacil- 

*  "Weekblad  van  het  Nederlandsch  Tijdschrift  voor  geneeskunde," 
Deel  n,  1898,  No.  14;  abstract  "Centralbl.  f.  Bakt.,"  etc.,  xxv,  p.  257, 
1899- 

t"Brit.  Med.  Jour.,"  Feb.  22,  1905,  and  "Indian  Med.  Gazette," 
1905. 


Cultivation  767 

lus  upon  media  free  from  sodium  chlorid.  The  technic  of  his 
method  is  thus  described  by  Rudolph*:  "Small  lumps  of 
pumice  stone  are  washed  and  then  dried  in  the  sun,  and  then 
allowed  to  absorb  a  mixture  of  i  ounce  of  meat  extract  and 
2  ounces  of  water.  This  pumice  stone  is  then  placed  in 
wide-mouthed  bottles  and  placed  in  the  autoclave.  Each 
bottle  is  provided  with  a  stopper  through  which  pass  two 
tubes,  the  one  tube  opening  into  the  autoclave  and  reaching 
nearly  to  the  bottom  of  the  bottle,  and  the  other  leading  from 
the  top  of  the  bottle  into  a  condenser  adjoining.  When  the 
cover  of  the  autoclave  is  adjusted  and  the  steam  admitted, 
then  in  the  case  of  each  bottle,  the  steam  passes  by  the  one 
tube  to  the  bottom  of  the  bottle,  and  rising  through  the 
pieces  of  pumice  stone,  the  steam,  carrying  with  it  the  volatile 
constituents  of  the  meat-extract,  reaches  the  condenser  by 
the  second  tube.  The  vapor  in  the  condenser  yields  the 
salt-free  nutrient  medium  in  the  proportion  of  2  liters  to 
each  ounce  of  meat-extract  originally  used.  The  medium  is 
collected  from  the  condenser  in  sterilized  Pasteur  flasks  which 
are  kept  plunged  during  the  process  in  a  freezing  mixture 
in  order  to  condense  some  of  the  volatile  alkaloids  from  the 
beef  that  would  otherwise  escape.  The  nutrient  fluid  is 
now  inoculated  with  the  bacillus  of  leprosy  and  the  flasks 
kept  at  37°  C.  for  from  four  to  six  weeks;  at  the  end  of  this 
period  when  examined  the  flasks  should  present  a  turbid 
appearance  with  a  stringy  white  deposit." 

Cleggt  announced  the  cultivation  of  lepra  bacilli  from 
human  leprous  tissue  in  symbiosis  with  ameba  and  other 
bacteria.  The  organisms  thus  cultured  he  kept  alive  in 
subcultures.  The  method  devised  by  Clegg  was  the  starting- 
point  of  a  more  extended  research  by  Duval,  J  who,  after  con- 
firming the  work  of  Clegg,  found  that  the  bacillus  could  be 
cultivated  directly  from  human  lesions  upon  culture-media 
containing  tryptophan,  without  the  symbiotic  ameba  or  other 
bacteria.  The  initial  culture  was  somewhat  difficult  to 
secure,  but  once  the  bacilli  grewT,  transplantation  was  easily 
and  successfully  carried  on  for  indefinite  generations.  He 
further  found  that  the  lepra  bacillus  could  be  successfully 
started  to  grow  upon  the  ordinary  laboratory  media  if  bits 
of  leprous  tissue  were  placed  upon  them,  and  at  the  same  time 

*  "Medicine,"  March,  1905,  p.  175. 

f  "Philippine  Journal  of  Science,"  1909,  iv,  403. 

J  "Journal  of  Experimental  Medicine,"  1910,  xn,  649;  1911,  xm,  365. 


768  Leprosy 

some  symbiotic  organism,  such  as  the  colon,  typhoid,  proteus, 
or  other  bacilli,  added.  Or  if  the  tissue  were  already  con- 
taminated the  lepra  bacilli  proceeded  to  multiply.  Duval 
interprets  this  to  mean  that  the  lepra  bacillus  is  unable  to 
effect  the  destruction  of  the  albumin  molecule  alone,  and  hence 
explains  the  advantage  of  adding  tryptophan.  The  medium 
most  successfully  employed  by  Duval  is  as  follows : 

"  Egg- albumen  or  numan  blood-serum  is  poured  into 
sterile  Petri  dishes  and  inspissated  for  three  hours  at  70°  C. 
The  excised  leprous  nodule  is  then  cut  into  thin  slices,  2  to  4 
mm.  in  breadth  and  0.5  to  i  mm.  in  thickness,  which  are 
distributed  over  the  surface  of  the  coagulated  albumin. 
By  means  of  a  pipet  the  medium  thus  seeded  with  bits  of 
tissue  is  bathed  in  a  i  per  cent,  sterile  solution  of  trypsin,  care 
being  taken  not  to  submerge  the  pieces  of  leprous  tissue. 
Sufficient  fluid  is  added  to  moisten  thoroughly  the  surface  of 
the  medium.  The  Petri  dishes  are  now  placed  in  a  moist 
chamber  at  37°  C.,  and  allowed  to  incubate  for  a  week  or  ten 
days.  They  are  removed  from  the  plates  from  time  to  time, 
as  evaporation  necessitates,  for  the  addition  of  more  trypsin. 
It  will  be  noted  that  after  a  week  or  ten  days  the  tissue  bits 
are  partially  sunken  below  the  surface  of  the  medium  and  are 
softened  to  a  thick,  creamy  consistence,  fragments  of  which 
are  readily  removed  with  a  platinum  needle.  On  microscopic 
examination  of  this  material  it  is  noted  that  the  leprosy  bacilli 
have  increased  to  enormous  numbers  and  scarcely  a  trace  of 
the  tissue  remains.  Separate  lepra  bacillus  colonies  are  also 
discernible  on  and  around  the  softened  tissue  masses.  .  .  . 
The  colonies  are  at  first  grayish  white,  but  after  several  days 
they  assume  a  distinct  orange-yellow  tint.  .  .  .  Subcultures 
may  be  obtained  by  transferring  portions  of  the  growth  to  a 
second  series  of  plates  or  to  slanted  culture- tubes  that  contain 
the  special  albumin- trypsin  medium.  After  the  third  or 
fourth  generation  the  bacilli  may  be  grown  without  difficulty 
upon  glycerinated  serum  agar  prepared  in  the  following 
manner : 

'  Twenty  grams  of  agar,  3  gm.  of  sodium  chlorid,  30  c.c. 
of  glycerin,  and  500  c.c.  of  distilled  water  are  thoroughly 
mixed,  clarified,  and  sterilized  in  the  usual  way.  To  tubes 
containing  10  c.c.  of  this  material  is  added  in  proper  propor- 
tion a  solution  of  unheated  turtle  muscle  infusion.  Five 
hundred  grams  of  turtle  muscle  are  cut  into  fine  pieces  and 
placed  in  a  flask  with  500  c.c.  of  distilled  water.  This  is 


Cultivation  769 

kept  in  the  ice-chest  for  forty-eight  hours  and  then  filtered 
through  gauze  to  remove  the  tissue.  The  filtrate  is  then 
passed  through  a  Berkefeld  filter  for  purposes  of  sterilization. 
By  means  of  a  sterile  pipet,  5  c.c.  of  the  muscle  filtrate  is 
added  to  the  agar  mixture  which  has  been  melted  and  cooled 
to  42°  C.  The  tubes  are  now  thoroughly  agitated  and 
allowed  to  solidify  in  the  slanted  position. 

"  This  medium  is  perfectly  clear  or  of  a  light  amber  color, 
and  admirably  suited  to  the  cultivation  of  the  Bacillus  lepra, 
once  the  initial  culture  has  been  started.  Growth  is  luxuriant 
and  reaches  its  maximum  in  forty -eight  to  sixty  hours.  On 
the  surface  of  this  medium  the  growth  is  moist  and  orange- 
yellow  in  color,  while  in  the  water  of  condensation,  though 
growth  apparently  has  not  occurred,  the  detached  bacilli  col- 
lect in  the  dependent  parts  in  the  form  of  feathery  masses 
without  clouding  the  fluid. 

"  Ordinary  nutrient  agar  may  be  used  with  trypsin  as  a 
plating  medium  instead  of  the  inspissated  serum  where  bits 
of  tissue  are  employed.  With  the  addition  of  i  per  cent,  of 
tryptophan  it  answers  every  purpose,  whether  the  bacilli  are 
planted  with  tissue  or  alone.  It  also  serves  to  start  multipli- 
cation of  lepra  bacilli  that  are  contaminated  at  the  time  of 
plating.  In  the  latter  case  the  medium  is  '  surface  seeded  ' 
with  an  emulsion  of  the  tissue  juices  in  the  same  manner  as 
in  preparing  '  streak  '  plates.  The  leprosy  colonies  in  the 
thinner  parts  of  the  loop  track  are  well  separated  and  easily 
distinguished  from  those  of  other  species  by  their  color  and 
by  their  appearance  only  after  two  to  five  days. 

"  In  using  an  agar  medium  it  is  well  to  leave  out  the  pep- 
tone and  to  titrate  the  reaction  to  1.5  per  cent,  alkaline  in 
order  to  prevent  too  profuse  growth  of  the  associated  bacteria ; 
besides,  an  alkaline  medium  seems  best  adapted  for  the  mul- 
tiplication of  the  lepra  bacillus. 

"  Bacillus  leprse  will  also  grow  on  the  various  blood-agar 
media  once  they  are  accustomed  to  artificial  conditions.  The 
Novy-McNeal  agar  for  the  cultivation  of  trypanosomes  gives 
a  luxuriant  growth  of  the  organism  if  2  per  cent,  glycerin  has 
been  added;  without  the  glycerin,  growth  is  very  scant. 
Fluid  media  are  not  suited  for  the  artificial  cultivation  of 
leprosy  bacilli  unless  they  are  kept  upon  the  surface.  Like 
the  tubercle  bacilli  they  require  abundant  oxygen.  .  .  . 

"  Ordinarily  the  growth  of  Bacillus  leprae  is  very  moist,  and 
in  this  respect  unlike  that  of  Bacillus  tuberculosis,  except 
49 


770  Leprosy 

possibly  the  avian  stain.  Sometimes  when  the  medium  is 
devoid  of  water  of  condensation,  the  growth  is  dry  and  oc- 
casionally wrinkled,  though  it  is  easily  removed  from  the 
surface  of  the  medium. 

"The  chromogenic  property  of  lepra  cultures  is  a  constant 
and  characteristic  feature  of  the  rapidly  growing  strains. 
The  color  varies  in  the  degree  of  intensity  depending  upon  the 
medium  employed.  If  glycerinated  agar  (without  peptone)  is 
used,  the  colonies  are  faint  lemon,  while  on  inspissated  blood- 
serum  they  are  deep  orange.  It  is  noteworthy  that  the 
growth  in  the  tissues  and  in  the  first  dozen  or  so  generations 
on  artificial  media  is  entirely  without  pigment." 

Pathogenesis. — Melcher  and  Ortmann*  introduced  frag- 
ments of  lepra  nodules  into  the  anterior  chambers  of  the 
eyes  of  rabbits,  and  observed  the  death  of  the  animals  after 
some  months,  with  what  they  considered  to  be  typical  lep- 
rous lesions  of  all  the  viscera,  especially  the  cecum ;  but  the 
recent  careful  experiments  of  Tashiroj  show  that  most  of  the 
lower  animals  are  entirely  insusceptible  to  infection  with  the 
lepra  bacillus,  and  that  when  they  are  inoculated  the  bacilli 
persistently  diminish  in  numbers  and  finally  disappear. 

Nicolle  |  found  it  possible  to  infect  monkeys  with  material 
rich  in  lepra  bacilli  taken  from  human  beings.  The  lesions 
appeared  only  after  an  incubation  period  that  was  in  some 
cases  prolonged  from  twenty-two  to  ninety-four  days.  The 
lesions  persisted  but  a  short  time  and  the  monkeys  recovered 
in  from  thirty  to  one  hundred  and  fifty  days. 

Clegg§  and  Sugai||  found  Japanese  dancing  mice  suscep- 
tible to  infection  with  leprous  material,  the  micro-organisms 
not  remaining  localized  at  the  seat  of  inoculation,  but  dissem- 
inating throughout  the  animal's  body.  Their  observation 
has  been  confirmed  by  Duval,**  who  later  ff  was  also  able  to 
infect  monkeys — Macacus  rhesus — with  pure  cultures  of  the 
organism  and  produce  the  typical  disease. 

Very  few  instances  are  recorded  in  which  actual  inocula- 

*  "Berliner  klin.  Wochenschrift,"  1885-1886. 

f  "Centralbl.  f.  Bakt.  u.  Parasitenk."  Originale),  xxxi,  No.  7,  p. 
276,  March  12,  1902. 

f  "Semaine  medicale,"  1905,  No.  10,  p.  no. 

§  "Philippine  Journal  of  Science,"  1909,  iv,  403. 

||  "Lepra,"  1909,  vin,  203. 

**  "Journal  of  Experimental  Medicine,"  1910,  xn,  649. 
tt  Ibid.,  1911,  xiu,  374. 


Lesions  771 

tion  has  produced  leprosy  in  man.  Arning*  was  able  to 
experiment  upon  a  condemned  criminal,  of  a  family  entirely 
free  from  the  disease,  in  the  Sandwich  Islands.  Fragments 
of  tissue  freshly  excised  from  a  lepra  nodule  were  introduced 
beneath  his  skin  and  the  man  was  kept  under  observation. 
In  the  course  of  some  months  typical  lesions  began  to 
develop  at  the  points  of  inoculation  and  spread  gradually, 
ending  in  general  leprosy  in  about  five  years. 

Sticker  |  is  of  the  opinion  that  the  primary  infection  in 
lepra  takes  place  through  the  nose,  supporting  his  opinion 
by  observations  upon  153  accurately  studied  cases,  in  which — 

1.  The  nasal  lesion  is  the  only  one  constant  in  both  the 
nodular  and  anesthetic  forms  of  the  disease. 

2.  The  nasal  lesion  is  peculiar — i.  e.,  characteristic — and 
entirely  different  from  all  other  lepra  lesions. 

3.  The  clinical  symptoms  of  lepra  begin  in  the  nose. 

4.  The  relapses  in  the  disease  always  begin  with  nasal 
symptoms,  such  as  epistaxis,  congestion  of  the  nasal  mucous 
membrane,  a  sensation  of  heat,  etc. 

5.  In  incipient  cases  the  lepra  bacilli  are  first  found  in 
the  nose. 

Lesions. — The  lepra  nodes  in  general  resemble  tubercu- 
lous lesions,  but  are  superficial,  affecting  the  skin  and  sub- 
cutaneous tissues.  Rarely  they  may  also  occur  in  the 
organs.  VirchowJ  has  seen  a  case  in  which  lepra  bacilli 
could  be  found  only  in  the  spleen. 

Once  established  in  the  body,  the  bacillus  may  grow  in 
the  connective  tissues  and  produce  chronic  inflammatory 
nodes — the  analogues  of  tubercles;  or  in  the  nerves,  causing 
anesthesia  and  trophic  disturbances.  On  this  account  two 
forms  of  the  disease — lepra  nodosa  (elephantiasis  graecorum) 
and  lepra  an&sthetica — are  described.  These  forms  may 
occur  independently  of  one  another,  or  may  be  associated 
in  the  same  case. 

The  nodes  consist  of  lymphoid  and  epithelioid  cells  and 
fibers,  and  are  vascular,  so  that  much  of  the  embryonal 
tissue  completes  its  transformation  to  fibers  without  necrotic 
changes.  This  makes  the  disease  productive  rather  than 
destructive,  the  lesions  resembling  new  growths.  The 

*  "Centralbl.  f.  Bakt.,"  etc.,  vi,  p.  201,  1889. 

t  "Mittheilungen    und  Verhandlungen  der  internationalen  wissen- 
schaftlichen  Lepra-Konferenz  zu  Berlin,"  Oct.,  1897,  2,  Theil. 
J  Ibid. 


772 


Leprosy 


bacilli,  which  occur  in  enormous  numbers,  are  often  found 
in  groups  inclosed  within  the  protoplasm  of  certain  large 
vacuolated  cells — the  "  lepra  cells  " — which  seem  to  be 
partly  degenerated  endothelial  cells.  Sometimes  they  are 
anuclear;  rarely  they  contain  several  nuclei  (giant  cells). 
Bacilli  also  occur  in  the  lymph-spaces  and  in  the  nerve-sheath. 


; 


Fig.  251. — Lepra  nervorum  (McConnell). 

Lepra  nodules  do  not  degenerate  like  tubercles,  and  the 
ulceration,  which  constitutes  a  large  part  of  the  pathology 
of  the  disease,  seems  to  be  largely  due  to  the  injurious  action 
of  external  agencies  upon  the  feebly  vital  pathologic  tissue. 

According  to  the  studies  of  Johnston  and  Jamieson,*  the 
bacteriologic  diagnosis  of  nodular  leprosy  can  be  made  by 
spreading  serum  obtained  by  scraping  a  leprous  nodule  upon 
a  cover-glass,  drying,  fixing,  and  staining  with  carbol- 
fuchsin  and  Gabbet's  solution  as  for  the  tubercle  bacillus. 


Montreal  Med.  Journal."  Jan.,  1897. 


Varieties 


773 


In  such  preparations  the  bacilli  are  present  in  enormous 
numbers,  thus  forming  a  marked  contrast  to  tuberculous 
skin  diseases,  in  which,  very  few  can  be  found. 

In  anesthetic  leprosy  nodules  form  upon  the  peripheral 
nerves,  and  by  connective-tissue  formation,  as  well  as  by 
the  entrance  of  the  bacilli  into  the  nerve-sheaths,  cause 
irritation,  followed  by  degeneration  of  the  nerves.  The 


Fig.  252. — A  case  of  lepra  nodosa  treated  in  the  Medico- Chirurgical 
Hospital  of  Philadelphia. 

anesthesia  following  the  peripheral  nervous  lesions  pre- 
disposes to  the  formation  of  ulcers,  etc.,  by  allowing  injuries 
to  occur  without  detection  and  to  progress  without  observa- 
tion. The  ulcerations  of  the  hands  and  feet,  with  frequent 
loss  of  fingers  and  toes,  follow  these  lesions,  probably  in  the 
same  manner  as  in  syringomyelia. 

The  disease  usually  first  manifests  itself  upon  the  face, 
extensor  surfaces,  elbows,  and  knees,  and  for  a  long  time 
confines  itself  to  the  skin.  Ultimately  it  sometimes  invades 


774  Leprosy 

the  lymphatics  and  extends  to  the  internal  viscera.  Death 
ultimately  occurs  from  exhaustion,  if  not  from  the  frequent 
intercurrent  affections,  especially  pneumonia  and  tuberculo- 
sis, to  which  the  conditions  predispose. 

Specific  Therapy. — Carrasquilla's*  "leprosy  serum"  is 
prepared  by  injecting  the  serum  separated  from  blood  with- 
drawn from  lepers,  into  horses,  mules,  and  asses,  and,  after 
a  number  of  injections,  bleeding  the  animals  and  sepa- 
rating the  serum.  There  is  no  reason  for  thinking  that  such 
a  product  could  have  therapeutic  value. 

Rostf  prepares  massive  cultures  of  the  lepra  bacillus, 
niters  them  through  porcelain,  concentrates  the  nitrate  to 
one- tenth  of  its  volume,  and  mixes  the  nitrate  with  an  equal 
volume  of  glycerin.  The  resulting  preparation  is  called 
leprolin  and  is  supposed  to  be  analogous  to  tuberculin. 
With  it  he  has  treated  a  number  of  lepers  at  the  Leper 
Hospital  at  Rangoon,  Burmah,  many  of  whom  have  greatly 
improved  and  some  of  whom  seem  to  be  cured.  Confirma- 
tion of  the  work  by  others  is  greatly  desired,  and  it  is  too 
early  to  judge  the  merits  of  the  treatment.  It  is,  however, 
the  most  promising  method  yet  published. 

Sanitation. — While  not  so  contagious  as  tuberculosis, 
it  has  been  proved  that  leprosy  is  transmissible,  and  it  may 
be  regarded  as  an  essential  sanitary  precaution  that  lepers 
should  be  segregated  and  mingle  as  little  as  possible  with 
healthy  persons.  The  disease  is  not  hereditary,  so  that 
there  is  no  reason  why  lepers  should  not  marry  among 
themselves.  The  children  should,  however,  be  taken  from 
the  parents  lest  they  be  subsequently  infected. 

*"  Wiener  med.  Wochenschrift,"  No.  41,  1897. 
f'Brit.  Med.  Jour.,"  Feb.  n,  1905. 


CHAPTER  XXIX. 

GLANDERS. 
BACILLUS  MALLEI  (LOFFLER  AND  SCHUTZ).* 

General  Characteristics. — A  non-motile,  non-flagellate,  non-spor- 
ogenous,  non-liquefying,  non-chromogenic,  non-aerogenic,  aerobic  and 
optionally  anaerobic,  acid-forming  and  milk  coagulating  bacillus,  patho- 
genic for  man  and  the  lower  animals,  staining  by  ordinary  methods, 
but  not  by  Gram's  method. 

Glanders  is  an  infectious  mycotic  disease  which,  for- 
tunately, is  almost  entirely  confined  to  the  lower  animals. 
Only  occasionally  does  it  secure  a  victim  among  hostlers, 
drovers,  soldiers,  and  others  whose  vocations  bring  them  in 
contact  with  diseased  horses.  Several  bacteriologists  have 
succumbed  to  accidental  laboratory  infection. 

Glanders  was  first  known  to  us  as  a  disease  of  the  horse 
and  ass,  characterized  by  the  formation  of  discrete,  cleanly 
cut  ulcers  upon  the  mucous  membrane  of  the  nose.  The 
ulcers  in  the  nose  of  the  horse  and  ass  are  formed  by  the 
breaking  down  of  inflammatory  nodules  which  can  be  detected 
in  all  stages  upon  the  diseased  membranes.  The  ulcers, 
having  once  formed,  show  no  tendency  to  recover,  but 
slowly  spread  and  persistently  discharge  a  virulent  pus. 
The  edges  of  the  ulcers  are  indurated  and  elevated,  their 
surfaces  often  smooth.  The  disease  does  not  progress  to 
any  great  extent  before  the  submaxillary  lymphatic  glands 
begin  to  enlarge,  soften,  open,  and  become  discharging 
ulcers.  The  lungs  may  also  become  infected  by  inspiration 
of  the  infectious  material  from  the  nose  and  throat,  and 
contain  small  foci  of  bronchopneumonia  not  unlike  tubercles 
in  their  early  appearance.  The  animals  ultimately  die  of 
exhaustion. 

Specific  Organism. — In  1882,  shortly  after  the  dis- 
covery of  the  tubercle  bacillus,  Loffler  and  Schiitz  discovered 
in  the  discharges  and  tissues  of  the  disease  the  specific 
micro-organism,  the  glanders  bacillus  (Bacillus  mallei). 

*"  Deutsche  med.  Wochenschrift,"  1882,  52. 
775 


776  Glanders 

Distribution. — The  glanders  bacillus  does  not  seem  to 
find  conditions  outside  the  animal  body  suitable  for  its 
growth,  and  probably  lives  a  purely  parasitic  existence. 

Morphology. — The  glanders  bacillus  is  somewhat  shorter 
and  distinctly  thicker  than  the  tubercle  bacillus,  and  has 
rounded  ends.  It  measures  about  0.25  to  0.4  X  1.5  to  3  [*, 
and  is  slightly  bent;  coccoid  and  branched  forms  sometimes 
occur.  It  usually  occurs  singly,  though  upon  blood-serum, 
and  especially  upon  potato,  conjoined  individuals  may  occa- 
sionally be  found.  Long  threads  are  never  formed. 


Fig-  253. — Bacillus   mallei,   from   a   culture  upon  glycerin  agar-agar. 
X  looo  (Frankel  and  Pfeiffer). 

When  stained  with  ordinary  aqueous  solutions  of  the 
aniline  dyes,  or  with  Loffler's  alkaline  methylene-blue,  the 
bacillary  substance  does  not  usually  appear  homogeneous, 
but,  like  that  of  the  diphtheria  bacillus,  shows  marked  in- 
equalities, some  area  being  deeply,  some  faintly,  stained. 

The  bacillus  is  non-motile,  has  no  flagella,  and  does  not 
form  spores. 

Staining. — The  organism  can  be  stained  with  the  watery 
anilin-dye  solutions,  but  not  by  Gram's  method.  The  bacil- 
lus readily  gives  up  the  stain  in  the  presence  of  decolorizing 
agents,  so  is  difficult  to  stain  in  tissues.  Loffler  accomplished 
the  staining  by  allowing  the  sections  to  lie  for  some  time 
(five  minutes)  in  the  alkaline  methylene-blue  solution,  then 


Isolation  777 

transferring  them  to  a  solution  of  sulphuric  and  oxalic 
acids — 

Concentrated  sulphuric  acid 2  drops 

Five  per  cent,  oxalic  acid  solution i  drop 

Distilled  water 10  c.c. 

for  five  seconds,  then  to  absolute  alcohol,  xylol,  etc.  The 
bacilli  appear  dark  blue  upon  a  paler  ground.  This  method 
gives  very  good  results,  but  has  been  largely  superseded  by 
the  use  of  Kiihne's  carbol-methylene-blue : 

Methylene-blue i  .5 

Alcohol 10.0 

Five  per  cent,  aqueous  phenol  solution 100.0 

Kiihne  stains  the  section  for  about  half  an  hour,  washes  it 
in  water,  decolorizes  it  carefully  in  hydrochloric  acid  (10 
drops  to  500  c.c.  of  water),  immerses  it  at  once  in  a  solution 
of  lithium  carbonate  (8  drops  of  a  saturated  solution  of  lithium 
carbonate  in  10  c.c.  of  water),  places  it  in  a  bath  of  distilled 
water  for  a  few  minutes,  dips  it  into  absolute  alcohol  col- 
ored with  a  little  methylene-blue,  dehydrates  it  in  anilin 
oil  containing  a  little  methylene-blue  in  solution,  washes 
it  in  pure  anilin  oil,  not  colored,  then  in  a  light  ethereal  oil, 
clears  it  in  xylol,  and  finally  mounts  it  in  balsam. 

Vital  Resistance. — The  organism  grows  only  between 
25°  and  42°  C.  It  is  killed  by  exposure  to  60°  C.  for  two 
hours,  or  to  75°  C.  for  one  hour.  Sunlight  kills  it  after 
twenty-four  hours'  exposure.  Thorough  drying  destroys  it  in 
a  short  time.  When  planted  upon  culture-media,  sealed,  and 
kept  cool  and  in  the  dark,  it  may  be  kept  alive  for  months 
and  even  years.  Exposure  to  i  per  cent,  carbolic  acid  de- 
stroys it  in  about  half  an  hour;  i :  1000  bichlorid  of  mercury 
solution,  in  about  fifteen  minutes.  According  to  Hiss  and 
Zinsser,  it  may  remain  alive  in  the  water  of  horse-troughs 
for  seventy  days. 

Isolation. — Attempts  at  the  isolation  of  the  glanders 
bacillus  from  infectious  discharges  by  the  usual  plate  method 
are  apt  to  fail,  on  account  of  the  presence  of  other  more 
rapidly  growing  organisms. 

The  best  method  of  isolation  seems  to  be  by  infecting  an 
animal  and  recovering  the  bacillus  from  its  tissues. 

The  guinea-pig,  being  a  highly  susceptible  as  well  as  a 
readily  procurable  animal,  is  appropriate  for  the  detection 


778  Glanders 

and  isolation  of  the  bacillus.  When  a  subcutaneous  inocu- 
lation of  some  of  the  infectious  pus  is  made,  a  tumefaction 
can  be  observed  in  guinea-pigs  in  from  four  to  five  days. 
Somewhat  later  this  tumefaction  changes  to  a  caseous 
nodule,  which  ruptures  and  leaves  a  chronic  superficial  ulcer 
with  irregular  margins.  The  lymph-glands  speedily  become 
invaded,  and  in  four  or  five  weeks  signs  of  general  infec- 
tion appear.  The  lymph-glands,  especially  of  the  inguinal 
region,  suppurate,  and  the  testicles  frequently  undergo  the 
same  process.  Later  the  joints  are  affected  with  a  suppura- 
tive  arthritis,  the  pus  from  which  contains  the  bacilli.  The 
animal  eventually  dies  of  exhaustion.  No  nasal  ulcers 
form  in  guinea-pigs. 

In  field-mice  the  disease  is  much  more  rapid,  no  local 
lesions  being  visible.  For  two  or  three  days  the  animal 
seems  unwell,  its  breathing  is  hurried,  it  sits  with  closed 
eyes  in  a  corner  of  the  cage,  and  finally,  without  any  other 
preliminaries,  tumbles  over  on  its  side,  dead. 

From  the  tissues  of  the  inoculated  animals  pure  cultures 
are  easily  made.  Perhaps  the  best  places  from  which  to 
secure  a  culture  are  the  softened  nodes  which  have  not 
ruptured,  or  the  joints. 

Diagnosis  of  Glanders. — Straus*  has  given  us  a  method 
which  is  of  great  use,  both  for  isolating  pure  cultures  of  the 
glanders  bacillus  and  for  making  a  diagnosis  of  the  disease. 
But  a  short  time  is  required.  The  material  suspected  to 
contain  the  glanders  bacillus  is  injected  into  the  peritoneal 
cavity  of  a  male  guinea-pig.  In  three  or  four  days  the 
disease  becomes  established  and  the  testicles  enlarge;  the 
skin  over  them  becomes  red  and  shining;  the  testicles 
themselves  begin  to  suppurate,  and  often  evacuate  through 
the  skin.  The  animal  dies  in  about  two  weeks.  If,  how- 
ever, it  be  killed  and  its  testicles  examined,  the  tunica 
vaginalis  testis  will  be  found  to  contain  pus,  and  some- 
times to  be  partially  obliterated  by  inflammatory  exudation. 
The  bacilli  are  present  in  this  pus,  and  can  be  secured  from  it 
in  pure  cultures. 

The  value  of  Straus'  method  has  been  somewhat  lessened 
by  the  discovery  by  Kutcher,f  that  a  new  bacillus,  which 
he  has  classed  among  the  pseudo- tubercle  bacilli,  produces 
a  similar  testicular  swelling  when  injected  into  the  abdominal 

*  "Compt.  rendu  Acad.  d.  Sciences,"  Paris,  cvm,  530. 
t  "Zeitschrift  fur  Hygiene,"  Bd.  xxi,  Heft  i,  Dec.  6,  1895. 


Cultivation  779 

cavity;  also  by  Levy  and  Steinmetz,*  who  found  that 
Staphylococcus  pyogenes  aureus  was  also  capable  of  provok- 
ing suppurative  orchitis.  However,  the  diagnosis  is  certain 
if  a  culture  of  the  glanders  bacillus  be  secured  from  the 
pus  in  the  scrotum. 

As  the  purulent  discharges  from  the  noses  of  horses  and 
other  large  animals  commonly  contain  very  few  bacilli, 
their  detection  by  the  use  of  the  guinea-pig  inoculation  is 
much  simplified. 

For  the  diagnosis  of  the  disease  in  living  animals,  sub- 
cutaneous injections  of  mallein  (q.  v.)  are  also  employed. 

McFadyenj  was  the  first  to  recommend  agglutination  of 
the  glanders  bacillus  by  the  serum  of  supposedly  infected 
animals  as  a  test  of  the  existence  of  glanders.  The  subject 
has  been  somewhat  extensively  tried  and  officially  adopted 
by  the  Prussian  government.  Moore  and  Taylor,  J  in  a  recent 
review  and  examination  of  the  test,  conclude  that  it  is  easier 
and  quite  as  accurate  as  the  mallein  method  and  is  applicable 
in  cases  where  fever  exists.  The  maximum  dilution  of 
normal  horse-serum  that  will  macroscopically  agglutinate 
glanders  bacilli  is  i :  500,  but  occurs  in  very  few  cases.  The 
maximum  agglutinative  power  of  the  serum  of  diseased  horses 
not  suffering  from  glanders  is  not  higher  than  that  of  normal 
serum.  The  diagnosis  is  usually  not  difficult  to  make,  but 
requires  much  care.  Cultures  of  the  glanders  bacillus  some- 
times unexpectedly  lose  their  ability  to  agglutinate. 

The  diagnosis  of  glanders  by  means  of  the  complement- 
fixation  method  has  been  tried  with  glittering  results  by 
Mohler  and  Eichhorn.§ 

Cultivation. — The  bacillus  is  an  aerobic  and  optionally 
anaerobic  organism,  and  can  be  grown  in  bouillon,  upon 
agar-agar,  better  upon  glycerin  agar-agar,  very  well  upon 
blood-serum,  and  quite  characteristically  upon  potato.  The 
optimum  temperature  is  37.5°  C. 

Colonies. — Upon  4  per  cent,  glycerin  agar-agar  plates 
the  colonies  appear  upon  the  second  day  as  whitish  or  pale 
yellow,  shining,  round  dots.  Under  the  microscope  they 
are  brownish  yellow,  thick  and  granular,  with  sharp  borders. 

Bouillon. — In  broth  cultures  the  glanders  bacillus  causes 

*  "Berliner  klin.  Wochenschrift,"  March  18,  1895,  No.  n. 
t  "Jour.  Comp.  Path,  and  Therap.,"  1896,  p.  322. 
J  "Jour.  Infectious  Diseases,"  iv,  1907,  p.  85,  supplement. 
§  "Report  of  the  Bureau  of  Animal  Industry,"  1910. 


780 


Glanders 


turbidity,  the  surface  of  the  culture  being  covered  by  a 
slimy  scum.  The  medium  becomes  brown  in  color. 

Gelatin  is  not  liquefied.  The  growth  upon  the  surface  is 
grayish  white  and  slimy,  never  abundant. 

Agar-agar. — Upon  agar-agar  and  glycerin  agar-agar  the 
growth  occurs  as  a  moist  shining  layer. 

Blood-serum. — Upon  blood-serum  the  growth  is  rather 
characteristic,  the  colonies  along  the  line  of  inoculation 
appearing  as  circumscribed,  clear,  transparent  drops,  which 
later  become  confluent  and  form  a  transparent  layer  un- 
accompanied by  liquefaction. 


Fig.  254. — Culture  of  glanders  bacillus  upon  cooked  potato  (Loffler). 

Potato. — The  most  characteristic  growth  is  upon  potato. 
It  first  appears  in  about  forty-eight  hours  as  a  transparent, 
honey-like,  yellowish  layer,  developing  only  at  incubation 
temperatures,  and  soon  becoming  reddish-brown  in  color. 
As  this  brown  color  of  the  colony  develops,  the  potato  for  a 
considerable  distance  around  it  becomes  greenish  brown. 
Bacillus  pyocyaneus  sometimes  produces  somewhat  the  same 
appearance. 

Milk. — In  litmus  milk  the  glanders  bacillus  produces  acid. 
A  firm  coagulum  forms  and  subsequently  separates  from 
the  clear  reddish  whey. 

Metabolic  Products. — The  organism  produces  acids  and 
curdling  ferments.  It  forms  no  indol,  no  liquefying  or  pro- 


Pathogenesis  781 

teolytic  ferments.  There  is  no  exotoxin.  All  the  poisonous 
substances  seem  to  be  endotoxins. 

Mallein. — Babes,*  Bonome,f  Pearson,  J  and  others  have 
prepared  a  substance,  mallein,  from  cultures  of  the  glanders 
bacillus,  and  have  employed  it  for  diagnostic  purposes.  It 
seems  to  be  useful  in  veterinary  medicine,  the  reaction  fol- 
lowing its  injection  into  glandered  animals  being  similar  to 
that  caused  by  the  injection  of  tuberculin  into  tuberculosis 
animals.  The  preparation  of  mallein  is  simple.  Cultures  of 
the  glanders  bacillus  are  grown  in  glycerin  bouillon  for  sev- 
eral weeks  and  killed  by  heat.  The  culture  is  then  filtered 
through  porcelain,  to  remove  the  dead  bacteria,  and  evapo- 
rated to  one-tenth  of  its  volume.  Before  use  the  mallein  is 
diluted  with  nine  times  its  volume  of  0.5  per  cent,  aqueous 
carbolic  acid  solution.  The  dose  for  diagnostic  purposes  is 
0.25  c.c.  for  the  horse.  It  has  also  been  prepared  from 
potato  cultures,  which  are  said  to  yield  a  stronger  product. 
The  agent  is  employed  exactly  like  tuberculin,  the  tem- 
perature being  taken  before  and  after  its  hypodermic  injec- 
tion. A  febrile  reaction  of  more  than  1.5°  C.  is  said  to  be 
indicative  of  the  disease. 

Pathogenesis. — That  the  bacillus  is  the  cause  of  glanders 
there  is  no  room  to  doubt,  as  LofHer  and  Schutz  have  suc- 
ceeded, by  the  inocultion  of  horses  and  asses,  in  producing 
the  well-known  disease. 

The  goat,  cat,  hog,  field-mouse,  wood-mouse,  marmot, 
rabbit,  guinea-pig,  and  hedgehog  all  appear  to  be  susceptible. 
Cattle,  house-mice,  white  mice,  rats,  and  birds  are  immune. 

Infection  may  take  place  through  the  mucous  membranes 
of  the  nose,  mouth,  or  alimentary  tract,  and  apparently 
without  pre-existing  demonstrable  lesions. 

The  disease  assumes  either  an  acute  form,  characterized  by 
destructive  necrosis  and  ulceration  of  the  mucous  membranes 
with  fever  and  prostration,  terminating  in  pneumonia,  or, 
as  is  more  frequent,  a  chronic  form  ("  farcy  "),  in  which  the 
lesions  of  the  mucous  membranes  are  less  destructive  and  in 
which  there  is  a  generalized  distribution  of  the  micro-organ- 
isms throughout  the  body,  with  resulting  more  or  less  wide- 
spread nodular  formations  (farcy-buds)  in  the  skin.  The 

*  "Archiv.  de  Med.  exp.  et  d'Anat.  patholog.,"  1892,  No.  4. 
t  "Deutsche  med  Woch.,"  1894,  Nos.  36  and  38,  pp.  703,  725,  and  744. 
t  "Jour,  of  Comp.  Med.  and  Vet.  Archiv.,"  Phila.,  xn,  1891,  pp. 
411-415. 


782 


Glanders 


acute  form  is  quickly  fatal,  death  sometimes  coming  on  in 
from  four  to  six  weeks ;  the  chronic  form  may  last  for  several 
years  and  end  in  complete  recovery. 

Lesions. — When  stained  in  sections  of  tissue  the  bacilli 
are  found  in  small  inflammatory  areas.  These  nodules  can 
be  seen  with  the  naked  eye  scattered  through  the  liver, 
kidney,  and  spleen  of  animals  dead  of  experimental  glanders. 
They  consist  principally  of  leukocytes,  but  also  contain 
numerous  epithelioid  cells.  As  is  the  case  with  tubercles, 


Fig-  255. — Pustular  eruption  of  acute  glanders  as  exhibited  on  the  day 
of  the  patient's  death,  twenty-eight  days  after  initial  chill  (Zeit). 

the  centers  of  the  nodules  are  prone  to  necrotic  changes,  but 
the  cells  show  marked  karyorrhexis,  and  the  tendency  is  more 
toward  colliquation  than  caseation.  The  typical  ulcera- 
tions  depend  upon  retrogressive  changes  occurring  upon 
mucous  surfaces,  the  breaking  down  of  the  nodules  per- 
mitting the  softened  material  to  escape.  At  times  the 
lesions  heal  with  the  formation  of  stellate  scars. 

Baumgarten*  regarded  the  histologic  lesions  of  glanders 
as  much  like  those  of  the  tubercle.     He  first  saw  epithe- 
*  "  Pathologische  Mykologie,"  Braunschweig,  1890. 


Pathogenesis  783 

lioid  cells  accumulate,  followed  by  the  invasion  of  leuko- 
cytes. Tedeschi*  was  not  able  to  confirm  Baumgarten's 
work,  but  found  the  primary  change  to  be  necrosis  of  the 
affected  tissue  followed  by  invasion  of  leukocytes.  The 
observations  of  Wright  |  are  in  accord  with  those  of  Tedeschi. 
He  first  saw  a  marked  degeneration  of  the  tissue,  and  then 
an  inflammatory  exudation,  amounting  in  some  cases  to 
actual  suppuration. 

Glanders   in   Human   Beings. — Human   beings   are   but 
rarely  infected.     The  disease  has,  however,  occurred  among 


Fig.  256. — Farcy  affecting  the  skin  of  the  shoulder  (Mohler  and 
Eichhorn,  in  Twenty-seventh  Annual  Report  of  the  Bureau  of  Animal 
Industry,  U.  S.  Department  of  Agriculture,  1910). 

those  in  frequent  contact  with  many  horses  and  among 
bacteriologists.  It  occurs  either  in  an  acute  form  in  which, 
from  whatever  primary  focus  may  have  been  its  starting- 
point,  the  distribution  of  micro-organisms  may  be  so  rapid 
as  to  induce  an  affection  with  skin  lesions  resembling  small- 
pox and  terminating  fatally  in  eight  or  ten  days. 

The  chronic  form  in  man  is  chiefly  confined  to  the  nasal 
and  laryngeal  mucosa.  It  is  commonly  mistaken  for  more 
simple  infections,  and  though  it  sometimes  shows  its  character 
by  generalizing,  it  not  infrequently  recovers. 

*  "Ziegler's  Beitrage  z.  path.  Anat.,"  Bd.  xm,  1893. 

t  "Journal  of  Experimental  Medicine,"  vol.  I,  No.  4,  p.  577. 


784 


Glanders 


Virulence. — The  organism  is  said  to  lose  virulence  if 
cultivated  for  many  generations  upon  artificial  media. 
While  this  is  true,  attempts  to  attenuate  fresh  cultures  by 
heat,  etc.,  have  usually  failed. 

Immunity. — Leo  has  pointed  out  that  white  rats,  which 
are  immune  to  the  disease,  may  be  made  susceptible  by 
feeding  with  phloridzin  and  causing  glycosuria. 

Babes  has  asserted  that  the  injection  of  mallein  into  sus- 
ceptible animals  will  immunize  them  against  glanders. 
Some  observers  claim  to  have  seen  good  therapeutic  results 


I 


Fig.  257. — Lesions  of  glanders  in  the  nasal  septum  of  a  horse  (Mohler 
and  Eichhorn,  in  Twenty-seventh  Annual  Report  of  tthe  Bureau  of 
Animal  Industry,  U.  S.  Department  of  Agriculture,  1910). 

follow  the  repeated  injection  of  mallein  in  small  doses. 
Others,  as  Chenot  and  Picq,*  find  blood-serum  from  im- 
mune animals  like  the  ox  to  be  curative  when  injected 
into  guinea-pigs  infected  with  glanders. 

Pseudoglanders  Bacillus. — A  bacillus  similar  in  its  tinc- 
torial and  cultural  peculiarities,  but  not  pathogenic  for  mice, 
guinea-pigs,  or  rabbits,  was  isolated  from  pus  by  Selter.  f  The 
organism  was  called  the  pseudoglanders  bacillus.  A  similar 
one  had  previously  been  described  by  JJabes.f 

*  " Compte-rendu  de  la  Soc.  de  Biol.,"  March  26,  1892. 

t  "Centralbl.  f.  Bakt.,"  etc.,  Feb.  18,  1902,  xxxv,  5,  p.  529. 

I  "Archiv.  de  med.  exp.  et  d'anat.  path.,"  1891. 


CHAPTER   XXX. 
RHINOSCLEROMA. 

BACILLUS  RHINOSCLEROMATIS  (VON  FRISCH  *) . 

General  Characteristics. — A  non-motile,  non-flagellate,  non- 
sporogenous,  non-chromogenic,  aerobic  and  optionally  anaerobic,  cap- 
sulated  bacillus,  pathogenic  for  man  and  identical  with  Bacillus  pneu- 
moniae  of  Friedlander,  except  that  it  stains  by  Gram's  method. 

A  peculiar  disease  of  the  nares,  characterized  by  the 
formation  of  circumscribed  nodular  tumors,  and  known 
as  rhinoscleroma,  is  occasionally  seen  in  Austria-Hungary, 
Italy,  and  some  parts  of  Germany.  A  few  cases  have  been 
observed  among  the  foreign-born  residents  of  the  United 
States.  The  nodular  masses  are  flattened,  may  be  discrete, 
isolated,  or  coalescent,  grow  with  great  slowness,  and  recur 
if  excised.  The  disease  commences  in  the  mucous  mem- 
brane and  the  adjoining  skin  of  the  nose,  and  spreads  to  the 
skin  in  the  immediate  neighborhood  by  a  slow  invasion, 
involving  the  upper  lip,  jaw,  hard  palate,  and  sometimes 
even  the  pharynx.  The  growths  are  without  evidences  of 
acute  inflammation,  do  not  ulcerate,  and  upon  microscopic 
examination  consist  of  an  infiltration  of  the  papillary  layer 
and  corium  of  the  skin,  with  round  cells  which  in  part  change 
to  fibrillar  tissue.  The  tumors  possess  a  well-developed 
lymph-vascular  system.  Sometimes  the  cells  undergo  hya- 
line degeneration. 

In  the  nodes  von  Frisch  discovered  bacilli  closely  re- 
sembling the  pneumobacillus  of  Friedlander,  both  in  mor- 
phology and  vegetation,  and,  like  it,  surrounded  by  a 
capsule.  The  only  differences  between  the  bacillus  of 
rhinoscleroma  and  Bacillus  pneumoniae  of  Friedlander  are 
that  the  former  stains  well  by  Gram's  method,  while  the 
latter  does  not;  that  the  former  is  rather  more  distinctly 
rod-shaped  than  the  latter,  and  more  often  shows  its  capsule 
in  culture  media. 

The  bacillus  can  be  cultivated,  and  cultures  in  all  media 

*  "Wiener  med.  Wochenschrift,"  1882,  32. 
50  785 


786  Rhinoscleroma 

resemble  those  of  the  bacillus  of  Friedlander  (q.  v.)  so  closely 
as  to  be  indistinguishable  from  it.  Even  when  inoculated 
into  animals  the  bacillus  behaves  much  like  Friedlander 's 
bacillus. 

Inoculation  has,  so  far,  failed  to  reproduce  the  disease 
either  in  man  or  in  the  lower  animals. 


,%•! 


Fig.  258. — Bacillus  rhinoscleromatis.     Pure  culture  on  glycerin  agar- 
agar.     Magnified  1000  diameters  (Migula). 

Pathogenesis. — The  bacillus  is  said  to  be  pathogenic  for 
man  only,  producing  granulomatous  formations  of  the  skin 
and  mucous  membranes  of  the  anterior  and  posterior  nares. 
These  vary  in  structure  according  to  age.  The  young  nodes 
consist  of  a  loose  fibrillar  tissue  composed  of  lymphocytes, 
fibroblasts,  and  fibers.  Some  of  the  cells  are  large  and  have  a 
clear  cytoplasm  and  are  known  as  the  cells  of  Mikulicz.  In 
and  between  them  the  bacilli  are  found  in  considerable  num- 
bers. The  older  lesions  consist  of  a  firm  sclerotic  cicatricial 
tissue. 


CHAPTER   XXXI. 

SYPHILIS. 

ALTHOUGH  syphilis  has  been  well  known  for  centuries,  its 
specific  cause  has  only  recently  been  discovered.  The  fact 
that  the  disease  had  not  been  successfully  communicated 
to  any  of  the  lower  animals  was  supposed  to  be  a  sufficient 
explanation  of  the  delay  in  recognizing  it.  Such  has  not, 
however,  proved  to  be  the  case,  for  in  spite  of  the  discovery 
by  Metschnikoff  and  Roux*  that  chimpanzees  could  be  suc- 
cessfully inoculated  with  virus  from  a  human  lesion,  and  the 
confirmation  of  their  work  by  Lassarf  and  others,  and  the 
additional  discovery  of  Metschnikoff  and  Roux,  |  that  it  is 
also  possible  to  infect  macaques  with  syphilis,  the  specific 
organism  was,  after  all,  discovered  for  the  first  time  in 
matter  secured  from  human  lesions. 

TREPONEMA    (SPIROCH^TA)     PALUDUM    (SCHAUDINN   AND 
HOFFMANN). 

General  Characteristics.— A  minute,  slender,  spiral  organism, 
closely  coiled,  flexible,  non-chromogenic,  non-aerogenic,  anaerobic,  non- 
liquefying,  motile,  flagellated,  cultivable  upon  specially  prepared  media, 
pathogenic  for  man  and  certain  of  the  lower  animals,  staining  by  cer- 
tain methods  only  and  not  by  Gram's  method.  • 

It  has  been  known  for  a  long  time  that  preputial  smegma 
and  various  ulcerative  lesions  of  the  generative  organs  con- 
tain certain  spiral  organisms.  Bordet  studied  these  with 
some  care,  expecting  to  prove  that  they  were  concerned 
with  the  etiology  of  syphilis,  but  it  remained  for  Schaudinn 
and  Hoffmann  §  to  point  out  that  there  were  two  separate 
species — one,  which  they  call  Spirochaeta  refringens,  com- 
monly found  in  ulcerative  lesions  of  the  genitalia,  and 
another,  called  Spirochaeta  pallida,  later,  and  more  correctly, 
Treponema  pallidum,  found  only  in  syphilitic  lesions — and, 

*  "Ann,  de  1'Inst.  Pasteur,"  Dec.,  1903,  p.  809. 
t  "Berliner  klin.  Wochenschrift,"  1903,  p.  1189. 
J  "Annales  de  1'Inst.  Pasteur,"  Jan.,   1904. 
§  "Deutsche  med.  Wochenschrift,"  May  4,  1905. 
787 


788  Syphilis 

therefore,  their  probable  cause.  The  observations  of  Schau- 
dinn  and  Hoffmann,  quickly  confirmed  by  Metschnikoff,* 
have  now  been  universally  accepted. 

Morphology. — The  organism  is  a  slender,  flexible,  closely 
coiled  spiral,  usually  showing  from  eight  to  ten  uniform  un- 
dulations, but  occasionally  being  so  short  as  to  show  only 
two  or  three,  or  so  long  as  to  show  as  many  as  twenty. 

It  is  very  slender,  measuring  from  0.33  to  0.5  [A  in  breadth 
to  3.5  to  15.5  11  in  length  (Levaditi  and  Mclntosh). 

It  forms  no  spores.  Multiplication  seems  to  take  place  by 
longitudinal  division. 

It  is  motile,  and  when  observed  alive  with  a  dark  field 
illuminator,  can  be  seen  to  rotate  slowly  about  its  longitu- 
dinal axis  at  the  same  time  that  it  slowly  sways  from  side 
to  side  with  a  serpentine  movement.  The  organisms  are 
provided  with  flagella  at  one  end,  sometimes  one  at  each 
end. 

Noguchif  observed  two  types  of  treponema,  one  slender, 
one  stouter.  When  carried  through  culture  and  used  to  in- 
oculate rabbits  their  differences  were  found  to  be  fairly  con- 
stant. The  lesions  produced  in  rabbit's  testicles  varied  with 
the  variety  of  organism  inoculated,  one  causing  a  diffuse,  the 
other  a  nodular,  orchitis.  He  conjectures  that  the  distinction 
may  be  of  value  in  explaining  certain  obscure  points  in  human 
syphilis. 

Staining. — The  original  discovery  of  the  organism  was 
achieved  through  the  employment  of  Giemsa's  stain — a 
modification  of  the  Romanowsky  method.  But  by  this 
method  the  organisms  appeared  very  pale  and  not  very 
numerous.  GoldhornJ  improved  the  method  as  follows: 

In  200  c.c.  of  water,  2  grams  of  lithium  carbonate  are  dissolved  and 
2  grams  of  Merck's  medicinal,  Griibler's  BX,  or  Koch's  rectified  methy- 
lene-blue  added.  This  mixture  is  heated  moderately  in  a  rice  boiler 
until  a  rich  polychrome  has  formed.  To  determine  this  a  sample  is 
examined  in  a  test-tube  every  few  minutes  by  holding  it  against  an 
artificial  light.  As  soon  as  a  distinctly  red  color  is  obtained,  the  desired 
degree  of  heating  has  been  reached.  After  cooling  it  is  filtered  through 
cotton  in  a  funnel.  To  one-half  of  this  polychrome  solution  5  per  cent, 
of  acetic  acid  is  gradually  added  until  a  strip  of  litmus-paper  shows 
above  the  line  of  demarcation  a  distinct  acid  reaction,  when  the  re- 
maining half  of  the  solution  is  added,  so  as  to  carry  the  reaction  back 
to  a  low  degree  of  alkalinity.  A  weak  eosin  solutioa  is  now  prepared, 

*  "Bull.  Acad.  de  med.  de  Paris,"  May  16,  1905. 

t  "Journal  of  Experimental  Medicine,"  1912,  xv,  No.  2,  p.  201. 

{Ibid.,  1906,  vin,  p.  451. 


Staining  789 


approximately  0.5  per  cent.  French  eosin,  and  this  is  added  gradually 
while  the  mixture  is  being  stirred  until  a  filtered  sample  shows  the  filtrate 
to  be  of  a  pale  bluish  color  with  a  slight  fluorescence.  The  mixture 
is  allowed  to  stand  for  one  day  and  then  filtered.  The  precipitate 
which  has  separated  is  collected  on  a  double  piece  of  filter-paper  and 
dried  at  room  temperature  (heating  spoils  it).  When  completely 
dried  it  can  easily  be  removed  from  the  paper  and  may  then  be  dissolved 
without  further  washing  in  commercial  (not  pure)  wood  alcohol.  The 
solution  should  be  allowed  to  stand  a  day,  then  filtered.  The  strength 
of  this  alcoholic  solution  is  approximately  i  per  cent.  To  use  the  stain, 
one  drops  upon  an  unfixed  spread  enough  dye  to  cover  it,  permits  it 
to  act  for  three  or  four  seconds,  and  then  pours  it  off  and  introduces  the 
glass  slowly,  spread  side  down,  into  clean  water,  where  it  is  held  for 
another  four  or  five  seconds,  after  which  it  is  shaken  to  and  fro  in  the 
water  to  wash  it.  It  is  next  dried  and  examined  at  once  or  after  mount- 
ing in  balsam.  The  spirochaetes  appear  violet  in  color. 

Ghoreyeb*  recommends  the  following  rapid  method  of 
staining  the  organism  in  smears.  A  thin  spread  is  to  be 
preferred.  No  heat  fixation  is  necessary: 

1.  Cover  the  smear  with  a  i  per  cent,  aqueous  solution  of  osmic  acid, 
and  permit  it  to  act  for  thirty  seconds.     This  solution  acts  as  a  fixative 
and  mordant. 

2.  Wash  thoroughly  in  running  water. 

3.  Cover  the  smear  with  a  i  :  100  dilution  of  Liquor  plumbi  subace- 
tatis  (freshly  prepared).     Permit  it  to  act  for  ten  seconds.      The  lead 
unites  with  the  albumin  to  form  lead  albuminate  which  is  insoluble  in 
water. 

4.  Wash  thoroughly  in  running  water. 

5.  Cover  the  smear  with  a  10  per  cent,  aqueous  solution  of  sodium 
sulphid.     This  is  to  act  ten  seconds,  during  which  the  salt  transforms 
the  lead  albuminate  into  lead  sulphid  and  causes  the  preparation  to  turn 
brown.     The  osmic  acid  when  reapplied  causes  it  to  become  black. 

6.  Wash  thoroughly  in  running  water. 

The  whole  process  is  to  be  repeated  in  exactly  the  same  man- 
ner three  times,  the  washings  all  being  very  thorough.  The 
preparation  is  then  dried  and  mounted  in  Canada  balsam. 
The  micro-organisms  and  cellular  detritus  are  stained  black. 

When  serum  from  a  primary  sore  or  other  syphilitic  lesion 
is  treated  by  these  methods,  a  number  of  the  spirochaeta  ap- 
pear well  stained  and  a  number  very  palely  stained,  so  that 
one  is  in  doubt  whether  there  may  be  many  others  un- 
stained, and  this  seems  to  be  the  case,  for  when  similar 
smears  are  treated  by  other  methods  many  more  can  be 
found. 

The  method  of  silver  incrustation  was  first  employed  for 
the  demonstration  of  the  organism  in  tissues,  but  Sternt 

"  Jour.  Amer.  Med.  Assoc.,"  May  7,  1910,  liv.,  No.  19,  p.  1498. 
t  "Berliner  klin.  Wochenschrift,"  1907,  No.  14. 


790 


Syphilis 


has  applied  it  with  great  success  to  the  examination  of 
fluids  by  the  following  simple  procedure:  Spreads  are  made 
in  the  usual  manner,  dried  in  the  air,  and  then  for  a  few 
hours  in  an  incubating  oven  at  37°  C.  They  are  next 
placed  in  a  10  per  cent,  solution  of  nitrate  of  silver  in  a 
colorless  glass  receptacle  and  allowed  to  rest  in  the  diffused 
daylight  of  a  comfortably  lighted  room  for  a  few  hours,  until 
they  become  brownish  metallic  in  appearance,  when  they 
are  thoroughly  washed  in  water.  The  spirochaeta  appear 
black,  the  background  brownish. 


Fig.  259. — Treponema  pallidum  in  the  periosteum  near  an  epiphysis 

(Bertarelli). 

Staining  the  organism  in  the  tissues  was  found  to  be  a  more 
difficult  matter,  for  the  Giemsa  stain  scarcely  showed  it  at  all. 
Bertarelli  and  Volpino*  endeavored  to  stain  sections  by  a 
modification  of  the  van  Ermengen  method  for  flagella  and 
had  some  success,  but  the  demonstration  of  the  organisms 
in  tissue  was  not  really  successful  until  Levaditif  devised 
the  method  of  silver  impregnation.  This  consists  in  hard- 
ening pieces  of  tissue  about  i  mm.  in  thickness  in  10  per 
cent,  formol  for  twenty-four  hours,  rinsing  in  water,  and 
immersing  in  95  per  cent,  alcohol  for  twenty-four  hours. 

'  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  Orig.,  1905,  XL,  p.  56. 
f  "Compt.-rendu  de  la  Soc.  de  Biol.  de  Paris,"  1905,  ux,  p.  326. 


Staining 


791 


The  block  is  then  placed  in  diluted  water  until  it  sinks  to 
the  bottom  of  the  container,  and  then  transferred  to  a 
1.5  to  3  per  cent,  aqueous  solution  of  nitrate  of  silver  in  a  blue 
or  amber  bottle  and  kept  in  a  dark  incubating  oven  at  37°  C. 
for  from  three  to  five  days.  Finally,  it  is  washed  in  water  and 
placed  in  a  solution  of  pyrogallic  acid  2  to  4  grams;  formol, 
5c.c. ;  distilled  water,  100  c.c.,  and  kept  in  the  dark,  at  room 
temperature,  from  twenty-four  to  seventy-two  hours,  then 


Fig.  260. — Treponema  pallidum  impregnated  with  silver.  Film 
prepared  from  the  skin  of  a  macerated,  congenitally  syphilitic  fetus. 
X  750  diameters  (Flexner).  The  dense  aggregation  of  organisms  may 
indicate  agglutination. 

washed  in  distilled  water,  embedded  in  paraffin,  and  cut. 
The  treponemata  are  intensely  black,  the  tissue  yellow  brown. 
The  sections  are  finally  stained  with — (a)  Giemsa's  stain  for 
a  few  minutes,  then  washed  in  water,  differentiated  with 
absolute  alcohol  containing  a  few  drops  of  oil  of  cloves, 
cleared  with  oil  of  bergamot  or  xylol,  or  (6)  concentrated 
solution  of  toluidin  blue,  differentiated  in  alcohol  containing 
a  few  drops  of  Unna's  glycerin-ether  mixture,  cleared  in  oil 
of  bergamot,  then  in  xylol,  and  mounted  in  Canada  balsam. 


792  Syphilis 

This  method  was  later  improved  by  Levaditi  and  Mamou- 
elian*  by  the  addition  of  10  per  cent,  of  pyridin  to  the 
silver  bath  just  before  the  block  of  tissue  is  put  in,  and 
by  using  for  the  reducing  bath  a  mixture  of  pyrogallic 
acid,  acetone,  and  pyridin.  The  details  are  as  follows: 
Fragments  of  organs  or  tissues  i  to  2  mm.  in  thickness  are 
fixed  for  twenty-four  to  forty-eight  hours  in  a  solution  of 
formalin  10  :  100,  then  washed  in  96  per  cent,  alcohol  for 
twelve  to  sixteen  hours,  then  in  distilled  water  until  the 
blocks  fall  to  the  bottom  of  the  container.  They  are 
then  impregnated  by  immersion  in  a  bath  composed  of 
a  i  per  cent,  solution  of  nitrate  of  silver,  to  which,  at  the 
moment  of  employment,  10  per  cent,  of  pyridin  is  added. 
Keep  the  blocks  immersed  in  this  solution  at  room  temper- 
ature for  two  or  three  hours,  and  at  50°  C.  for  four  or  six 
hours,  then  wash  rapidly  in  a  10  per  cent,  solution  of  py- 
ridin, and  reduce  in  a  bath  composed  of  4  per  cent,  pyro- 
gallic acid,  to  which,  at  the  moment  of  using,  10  per  cent,  of 
pure  acetone  and  15  per  cent,  (total  volume)  of  pyridin  are 
added.  The  reduction  bath  must  be  continued  for  several 
hours,  after  which  the  tissue  goes  through  70  per  cent, 
alcohol,  xylol,  paraffin,  and  sections  are  cut.  The  sections, 
fastened  to  the  slide,  are  stained  with  Unna's  blue  or  tolu- 
idin  blue,  differentiated  with  glycerin-ether,  and  finally 
mounted  in  Canada  balsam. 

Burrif  has  recommended  a  simple  and  rapid  method  of 
demonstrating  the  treponema  and  other  similar  organisms  by 
the  use  of  India  ink.  A  drop  of  juice  is  squeezed  from  a 
chancre  or  mucous  patch  and  mixed  with  a  drop  of  India  ink 
and  then  spread  upon  a  glass  slide  as  in  making  a  spread  of 
a  drop  of  blood.  As  the  ink  dries  it  leaves  a  black  or  dark 
brown  field  upon  which  the  spiral  organisms  stand  out  as 
shining,  colorless,  and  hence  conspicuous  objects.  Williams 
uses  Higgins'  water-proof  ink,  and  Hiss  recommends  "chin- 
chin,"  Giinther- Wagner  liquid  pearl  ink  for  the  purpose. 

The  method  is  fairly  satisfactory  for  diagnosis  and  can  be 
applied  in  a  few  moments. 

Distribution. — The  Treponema  pallidum  is  not  known  in 
nature  apart  from  the  lesions  of  syphilis.  It  has  now  been 
found  in  all  the  lesions  of  this  disease  and  in  the  blood  of 
syphilitics  in  larger  or  smaller  numbers.  The  discovery  has 

*  "Compt.-rendu  de  la  Soc.  de  Biol.  de  Paris,"  1906.  LVIII,  p.  134. 
f  "Wiener  klin.  Wochenschrift,"  July  i,  1909. 


Cultivation  793 

greatly  modified  our  ideas  of  the  tertiary  stage,  for  the 
demonstration  of  the  organisms  in  its  lesions  shows  them  to 
be  undoubtedly  contagious.  The  greatest  number  of  the 
organisms  are  found  in  the  tissues — especially  the  liver — of 
still-born  infants  with  congenital  syphilis. 

Cultivation. — The  cultivation  of  the  treponema  was  first 
attempted  by  Levaditi  and  Mclntosh,*  who,  deriving  the 
organism  from  an  experimental  primary  lesion  in  a  monkey 
(Macacus  rhesus),  carried  it  through  several  generations 
in  collodion  sacs  inclosed  in  the  peritoneal  cavity  of  other 
monkeys  (Macacus  cynomolgus)  and  in  the  peritoneal 
cavity  of  rabbits.  They  were  unable,  however,  to  secure 
the  treponema  in  pure  culture,  having  it  continually  mixed 
with  other  organisms  from  the  primary  lesion.  In  the 
mixture,  however,  they  were  able  to  cultivate  it  for  genera- 
tions and  study  its  morphology  and  behavior.  During 
cultivation  its  virulence  was  lost. 

Schereschewsky  f  endeavored  to  cultivate  the  treponema 
by  placing  a  fragment  of  human  tissue  containing  it  deep 
down  into  a  high  layer  of  gelatinized  horse -serum.  The 
treponema  grew  together  with  the  contaminating  organism, 
and  no  pure  culture  was  secured.  Muhlens  J  and  Hoffmann,  § 
using  the  same  method,  succeeded  in  securing  pure  cultures 
of  the  treponema,  but  found  them  avirulent. 

Noguchi,||  taking  advantage  of  the  observations  of  Bruck- 
ner and  Galasesco  **  and  Sowade,  ft  that  an  enormous  multi- 
plication of  treponema  occurred  when  material  containing  it 
was  inoculated  into  the  rabbit's  testis,  performed  a  lengthy 
series  of  cultivation  experiments  with  the  enriched  material 
thus  obtained.  The  only  suitable  culture-medium  in  these 
earlier  experiments  proved  to  be  a  "  serum  water,"  composed 
of  i  part  of  the  serum  of  the  sheep,  horse,  or  rabbit  and  3  parts 
of  distilled  water;  16  c.c.  of  this  mixture  was  placed  in  test- 
tubes  20  cm.  long  and  1.5  cm.  in  diameter  and  sterilized  for 
fifteen  minutes  at  100°  C.  each  day  for  three  days. 

To  each  of  a  series  of  such  tubes  a  carefully  removed  frag- 

*  "Ann.  de  1'Inst.  Pasteur,"  1907,  p.  784. 

t" Deutsche  med.  Wochenschrift,"  1909,  xxxv,  835,  1260,  1652. 

t  Ibid.,  1909,  xxxv,  1261. 

§  "Zeitschrift  fur  Hygiene  und  Infektionsk.,"  1911,  LXVIII,  27. 

||  "Journal  of  Experimental  Medicine,"  1911,  xiv,  99. 
**  "Compt.-rendu  de  la  Soc.  de  Biol.  de  Paris,"  1910,  LXVIII,  648. 
ft  "Deutsche  med.  Wochenschrift,"  1911,  xxxvii,  682. 


794  Syphilis 

ment  of  sterile  rabbit's  testis  was  added,  after  which  the  tubes 
were  incubated  at  37°  C.  for  two  days  to  determine  their 
sterility.  To  each  tube  the  material  from  the  inoculated 
rabbit's  testis,  rich  in  the  treponema,  is  added,  after  which  the 
surface  of  the  medium  in  each  receives  a  thick  layer  of  sterile 
paraffin  oil.  As  the  most  strict  anaerobiosis  is  necessary,  the 
tubes  are  placed  in  a  Novy  jar,  the  bottom  of  which  contains 
pyrogallic  acid.  Noguchi  first  passes  H  gas  through  the 
jar,  permitting  it  to  bubble  through  the  pyrogallic  acid  solu- 
tion for  ten  minutes.  He  then  uses  a  vacuum  pump  to  ex- 
haust the  atmosphere  in  the  jar,  and  lastly  permits  the  alka- 
line solution  (KOH)  to  flow  down  one  of  the  tubes  to  mix  with 
the  pyrogallic  acid. 

In  these  cultures  the  pallidum  grows  together  with  such 
bacteria  as  may  have  been  simultaneously  introduced.  To 
secure  the  cultures  free  from  these  bacteria  Noguchi  permitted 
the  treponema  to  grow  through  a  Berkefeld  filter,  which  for 
a  long  time  held  back  the  other  organisms.  Later  it  was  found 
that  both  bacteria  and  treponema  grow  side  by  side  in  a  deep 
stab  in  a  serum-agar-tissue  medium.  Under  these  conditions 
the  bacteria  grow  only  in  the  stab  or  puncture,  but  the  trepo- 
nema grow  out  into  the  medium  as  a  hazy  cloud.  By  cau- 
tiously breaking  the  tube  and  securing  material  for  transplan- 
tation from  such  a  scarcely  visible  cloud,  the  organisms  may 
be  transplanted  from  the  new  media  and  pure  cultures  thus 
obtained. 

In  a  later  paper,  Noguchi*  details  the  cultivation  of  the 
treponema  from  fragments  of  human  chancres,  mucous 
patches,  and  other  cutaneous  lesions.  The  medium  employed 
is  a  mixture  of  2  per  cent,  slightly  alkaline  agar  and  i  part  of 
ascitic  or  hydrocele  fluid,  at  the  bottom  of  which  a  fragment 
of  rabbit  kidney  or  testis  is  placed.  The  medium  is  prepared 
in  the  tubes,  after  the  addition  of  the  tissue,  by  mixing  2 
parts  of  the  melted  agar  at  50°  C.  with  i  part  of  the  ascitic 
or  hydrocele  fluid.  After  solidification  a  layer  of  paraffin  oil 
3  cm.  deep  is  added. 

A  considerable  number  of  tubes  should  be  prepared  at  the 
same  time  and  incubated  for  a  few  days  prior  to  use  to  deter- 
mine sterility.  The  bits  of  human  tissue  are  snipped  up  with 
sterile  scissors  in  salt  solution  containing  i  per  cent,  of  sodium 
citrate  and  should  be  kept  immersed  in  this  fluid  from  the  time 
of  securing  to  the  time  of  planting,  so  as  not  to  become  dried. 
*  "Journal  of  Experimental  Medicine,"  1912,  xv,  i,  p.  90. 


Pathogenesis  and  Specificity  795 

A  bit  of  the  tissue  should  be  emulsified  in  a  mortar  with  citrate 
solution  and  examined  with  a  dark  field  illuminator  to  make 
sure  that  the  organisms  to  be  cultivated  are  present. 

If  they  are  found,  and  the  material  shown  to  be  adapted 
to  cultivation,  each  of  the  remaining  bits  of  tissue  is  taken  up 
by  a  thin  blunt  glass  rod  and  pushed  to  the  bottom  of  a  cul- 
ture-tube and  into  each  tube  several  drops  of  the  emulsion 
examined  are  introduced  by  means  of  a  capillary  pipet,  also 
inserted  deeply  into  the  medium.  The  tubes  are  next  incu- 
bated at  37°  C.  for  two  or  three  weeks.  In  successful  tubes, 
in  which  the  medium  has  not  been  broken  up  by  gas-producing 
bacteria,  there  is  a  dense  opaque  growth  of  bacteria  along  the 
line  of  puncture,  and  a  diffuse  opalescence  of  the  agar-agar 
caused  by  the  extension  into  it  of  the  growing  treponema. 
A  capillary  tube  cautiously  inserted  into  the  opalescent  me- 
dium withdraws  a  particle  that  can  be  examined  with  dark  field 
illuminator.  When  such  observation  shows  the  cause  of  the 
opalescence  to  be,  in  fact,  the  treponema,  the  tube  can  be 
cautiously  broken  at  some  appropriate  part  and  the  trans- 
plantation made  from  the  opalescent  part  of  the  medium  to 
fresh  appropriate  culture-media.  By  these  means,  after  a  few 
trials,  pure  cultures  of  treponema  were  secured. 

The  colonies  were  said  never  to  be  sharp,  but  always  faintly 
visible.  There  is  no  color  and  no  odor. 

By  inoculating  the  organisms  recently  secured  from  human 
lesions  (by  the  method  given)  into  monkeys  (Macacus  rhesus 
and  Cereopithicus  callitrichus)  Noguchi  was  able  to  produce 
typical  syphilis  of  the  monkey,  thus  showing  that  the  virulence 
of  the  organisms  was  not  lost  in  the  cultivation. 

Pathogenesis  and  Specificity. — There  can  be  no  doubt 
about  the  causal  relation  of  Treponema  pallidum  to  syphilis. 
It  is  unknown  in  every  other  relation;  it  has  appeared  in 
every  required  relation,  and  thus  has  completely  fulfilled  the 
laws  of  specificity  laid  down  by  Koch.  Treponema  pallidum 
is  not  only  pathogenic  for  man,  but,  as  has  already  been 
shown,  can  also  be  successfully  implanted  into  chimpanzees, 
macaques,  rabbits,  guinea-pigs,  and  other  experiment  animals. 
As  syphilis  is,  however,  unknown  under  natural  conditions, 
except  in  man,  it  may  be  looked  upon  as  a  human  disease. 

The  organism  enters  the  body  through  a  local  breach  of 
continuity  of  the  superficial  tissues,  except  in  experimental 
and  congenital  infections,  where  it  may  immediately  reach 
the  blood. 


796  Syphilis 

In  ordinary  acquired  syphilis  the  point  of  entrance  shows 
the  first  manifestations  of.  the  disease  after  a  period  of  pri- 
mary incubation  about  three  weeks  long,  in  what  is  known 
as  the  primary  lesion  or  chancre.  This  appears  as  a  papule, 
grows  larger,  undergoes  superficial  indolent  ulceration, 
and  eventually  heals  with  the  formation  of  an  indurated 
cicatrix.  It  is  in  this  lesion  that  the  treponema  first  makes 
its  appearance.  From  this  lesion,  where  it  multiplies 
slowly,  it  enters  the  lymphatics  and  soon  reaches  the  lymph- 
nodes,  which  swell  one  by  one  as  its  invasion  progresses. 
During  this  stage  of  glandular  enlargement  the  organisms 
can  be  found  in  small  numbers  in  juice  secured  from  a  punc- 
ture made  in  the  gland  with  a  hollow  needle.  This  period 
of  primary  symptoms  (chancre  and  adenitis)  includes  part 
of  what  is  known  as  the  period  of  secondary  incubation, 
which  intervenes  between  the  appearance  of  the  chancre  and 
that  of  the  secondary  symptoms.  It  usually  lasts  about  six 
weeks.  During  this  time  the  organisms  are  multiplying 
in  the  lymph-nodes  and  occasionally  entering  the  blood. 
What  fate  the  organisms  meet  when  they  reach  the  blood 
in  small  numbers  is  not  yet  known,  but  the  slow  inva- 
sion suggests  that  those  first  entering  are  destroyed,  and  that 
it  is  only  when  their  numbers  are  great  and  their  viru- 
lence increased  that  they  suddenly  become  able  to  over- 
come the  defenses  and  permit  the  development  of  the 
secondary  symptoms.  This  period  of  secondary  symptoms 
corresponds  to  the  invasion  of  the  blood  by  the  parasite. 
It  may  continue  from  one  to  three  years,  during  which  time 
the  patient  suffers  from  general  symptoms,  fever,  etc.,  proba- 
bly due  to  intoxication  and  local  symptoms,  such  as  alopecia, 
exanthemata,  etc.,  due  to  local  colonization  of  the  organ- 
isms. At  the  end  of  this  period  a  partial  immunity,  such  as 
is  seen  in  other  infectious  diseases  (malaria),  develops,  the 
organisms  disappear  from  the  blood,  the  general  local  and 
constitutional  disturbances  recover,  and  the  patient  may 
be  well.  Should  he  continue  to  harbor  some  of  the  micro- 
parasites,  however,  there  may  be  an  insidious  sclerosis  of 
the  blood-vessels  and  parenchymatous  organs  consequent 
upon  the  growth  and  multiplication  of  the  parasites,  or 
there  may  be  after  many  years  a  period  of  tertiary  symptoms 
characterized  by  the  sudden  appearance  of  severe  lesions 
in  which  the  parasites  are  very  few  in  number. 

The    specific   organisms    are    present   in    juice   expressed 


Diagnosis  797 

from  the  primary  lesion,  in  juice  from  the  bubos  and  en- 
larged lymph-nodes;  in  the  blood,  in  the  roseola,  and  all  of 
the  secondary  lesions,  and  sparingly  in  the  tertiary  lesions. 

In  congenital  syphilis  they  reach  the  fetus  from  either  the 
ovum,  the  spermatozoon,  or  from  the  blood  of  the  mother. 
Prenatal  death  from  syphilis  is  accompanied  by  lesions  in 
which  enormous  numbers  of  the  organisms  can  be  found, 
and  furnishes  the  best  tissues  for  their  experimental  demon- 
stration and  study. 

Lesions. — The  lesions  of  syphilis  are  so  numerous  that 
the  reader  is  referred  to  works  on  pathology  and  dermatology 
for  satisfactory  descriptions.  Here  it  may  suffice  to  say 
that  though  diverse  in  appearance  and  location,  they  have 
certain  features  in  common.  The  first  of  these,  and  that 
which  naturally  places  syphilis  among  the  infectious  granu- 
lomata,  is  the  lymphocytic  infiltration  of  the  tissues,  with 
which  all  of  the  lesions  begin.  The  second  is  a  peculiar 
form  of  necrosis — slimy  when  superficial,  gummy  when 
deep — with  which  they  terminate.  The  third  is  a  tendency 
toward  excessive  cicatrization. 

Diagnosis. — It  is  now  possible  to  make  a  certain  and 
early  diagnosis  of  syphilis  by  the  recognition  of  the  specific 
organisms,  and  as  the  difficulty  of  treatment  is  in  proportion 
to  the  stage  at  which  the  disease  arrives  before  treatment, 
it  should  never  be  neglected. 

I.  Staining. — The    expressed    lymph    from    a    carefully 
cleaned  freshly  abraded  primary  lesion  can  be  stained  by 
Giemsa's  method,  or,  as  is  much  better  and  more  certain,  by 
Stern's  method,  with  nitrate  of  silver,  or  by  the  use  of  India 
ink. 

II.  Dark-field  Examination. — For  those  who  possess  the 
"  dark-field  illuminator  "   or  some   similar   apparatus  with 
the  proper  lamp,  direct  examination  of  the  fluid  expressed 
from  the  lesions  can  be  made,  and  the  living,  moving  organ- 
isms recognized.     This  should  be  the  quickest  method  of 
diagnosis,  though  it  takes  practice. 

III.  Serum    Diagnosis. — Wassermann    and    Bruck    have 
devised  a  laboratory  method  of  making  the  diagnosis  of 
syphilis  by   testing    the    complement  fixing  power   of    the 
patient's  serum.     This  method,  now  known  as  the   "  Was- 
sermann reaction,"  is  given  in  complete  detail  under  a  more 
appropriate  heading.     (See  Wassermann  Reaction.) 

The  success  of  the  von  Pirquet  cutaneous  tuberculin  re- 


798  Syphilis 

action  in  assisting  the  diagnosis  of  tuberculosis  led  to  ex- 
periments on  the  part  of  a  number  of  investigators — Mei- 
rowsky,  Wolff-Eisner,  Tedeschi,  Nobe,  Ciuffo,  Nicholas, 
Favre,  and  Gauthier  and  Jodasshon — to  obtain  analogous 
reaction  in  syphilis  by  applying  extracts  of  syphilitic  tissues 
to  the  scarified  epiderm  of  syphilitics.  Some  reactions  were 
observed,  but  Neisser  and  Bruck  found  that  similar  reactions 
occurred  when  a  concentrated  extract  of  normal  liver  was 
applied,  and  to  such  reactions  which  could  not  be  looked  upon 
as  specific,  Neisser  applied  the  term  "  Umstimmung." 

After  having  successfully  achieved  the  cultivation  of  Tre- 
ponema  pallidum,  Noguchi*  resolved  to  try  the  effect  of  an 
application  of  an  extract  of  the  organisms  applied  to  the  skin, 
in  the  hope  that  it  might  provoke  a  reaction  useful  for  diag- 
nosis. To  this  end  he  prepared  two  cultures,  one  in  ascitic 
fluid  containing  a  piece  of  sterile  placenta,  the  other  in  ascitic 
fluid  agar  also  containing  a  piece  of  placenta.  After  per- 
mitting them  to  grow  under  strictly  anaerobic  conditions  at 
37°  C.  until  luxurient  development  occurred,  the  lower  part 
of  the  solid  culture  was  carefully  cut  off,  the  tissue  fragment 
removed,  and  the  rich  culture  carefully  ground  in  a  sterile 
mortar,  the  thick  paste  being  diluted  from  time  to  time  by 
adding  a  little  of  the  fluid  culture.  The  grinding  was  con- 
tinued until  the  emulsion  became  perfectly  clear,  when  it 
was  heated  to  60°  C.  for  one  hour  upon  a  water-bath  and  0.5 
per  cent,  of  carbolic  acid  added.  When  examined  with  the 
dark-field  illuminator,  40  to  100  dead  treponemata  could  be 
seen  in  every  field.  Cultures  made  from  the  suspension  re- 
mained sterile  and  inoculation  into  rabbits'  testicles  was  with- 
out result. 

This  extract  of  the  treponema  cultures  he  calls  luetin. 
When  it  was  applied  to  the  ear  of  a  normal  rabbit,  by  means  of 
an  endermic  injection  with  a  fine  needle,  an  erythema  ap- 
peared, but  faded  within  forty-eight  hours,  the  skin  resuming 
its  normal  appearance,  but  when  it  was  applied  to  the  ear  of  a 
syphilized  rabbit,  at  the  end  of  the  forty-eight  hours  the  red- 
ness developed  into  an  induration  the  size  of  a  pea  and  per- 
sisted from  four  to  six  days,  disappearing  in  ten  days.  In  one 
case  a  sterile  pustule  developed. 

Luetin  was  tested  by  Noguchi  and  his  colleagues  upon  400 
cases:  146  of  these  were  controls,  177  syphilitics,  and  77 
parasyphilitics.  In  the  controls  there  was  erythema  without 
*  "Journal  of  Experimental  Medicine,"  1911,  xm,  p.  557. 


Spirochaeta  Refringens  799 

pain  or  itching,  which  disappeared  without  induration  within 
forty-eight  hours.  In  the  syphilitics  at  the  end  of  forty-eight 
hours  there  was  an  induration  in  the  form  of  a  papule  5  to 
10  mm.  in  diameter,  surrounded  by  a  zone  of  redness  and 
telangiectasis.  This  slowly  increased  for  three  or  four  days 
and  became  dark  bluish  red.  It  usually  disappeared  in  about 
a  week.  Sometimes  the  papule  underwent  vesiculation  and 
sometimes  pustulation.  It  always  healed  kindly  without  in- 
duration. In  certain  cases  described  as  torpid,  the  erythema 
cleared  away  and  a  negative  result  was  supposed  to  have  re- 
sulted, when  suddenly  the  spots  lighted  up  again  and  pro- 
gressed to  vesiculation  or  pustulation.  In  3  cases  there  were 
constitutional  symptoms — malaise,  loss  of  appetite,  and  diar- 
rhea. Noguchi  found  that  the  reaction  is  specific,  that  it  is 
most  striking  and  most  constantly  present  in  tertiary, 
latent  tertiary,  and  congenital  syphilis.  It,  therefore,  forms 
a  valuable  adjunct  to  diagnosis,  seeing  that  it  is  most  evident 
in  precisely  those  cases  in  which  the  Wassermann  reaction  is 
most  apt  to  fail.  A  few  early  cases  energetically  treated  with 
mercury  and  salvarsan  give  marked  reactions.  A  few  old 
cases  fail  to  give  it. 

SPIROCH^TA   REFRINGENS    (SCHAUDINN  AND   HOFFMANN). 

This  spiral  organism,  though  given  the  name  by  which  it  is 
now  known  by  Schaudinn  and  Hoffmann,  was  probably  first 
described  by  Donne  under  the  name  Vibrio  lineola.  It  is 
probably  a  frequent  organism  of  the  skin  and  mucous  mem- 
branes, and  occurs  in  greatest  numbers  in  lesions  of  the  geni- 
talia  because  of  the  smegma  upon  which  it  customarily  lives. 
It  is  present  in  most  primary  lesions  of  syphilis,  but  is  no  less 
frequently  found  in  non-syphilitic  lesions,  such  as  balanitis, 
venereal  warts,  and  genital  carcinoma.  It  is  also  found  in  the 
mouth  and  on  the  tonsils.  According  to  Hoffmann  and 
Prowazek*  it  is  not  entirely  harmless,  but  has  a  pathogenic 
action,  and  some  of  the  complicating  lesions  of  syphilis  as 
well  as  some  of  the  destructive  diseases  of  the  genitals  may  be 
intensified  by  it.  They  found  it  pathogenic  for  apes. 

Morphologically,  it  is  much  broader  than  Treponema  palli- 
dum,  its  spiral  waves  are  much  coarser  and  less  regular.  It 
is  easy  to  stain  by  all  methods  and  is  hence  easily  found.  It 
has  been  cultivated  by  Noguchi. f 

*  "Centralbl.  f.  Bakt.,"  etc.,  1906,  xu. 

t  "Journal  of  Experimental  Medicine,"  May  i,  1912,  xv. 


CHAPTER   XXXII. 
FRAMBESIA  TROPICA  (YAWS). 

TREPONEMA  PERTENUE  (CASTELLANI). 

THIS  peculiar,  specific,  infectious,  contagious,  chronic  fe- 
brile disease  of  the  tropics  is  characterized  by  the  appearance 
of  one  or  more  primary  papular  lesions — the  yaws — bearing 
some  resemblance  to  raspberries,  upon  the  skin,  malaise, 
fever,  and  other  constitutional  disturbances.  These  are 
later  followed  by  the  appearance  of  a  second  crop  of  small 
papules  which  grow  to  the  size  of  a  pea  or  a  small  nut  or  may 
grow  to  be  as  large  as  apples,  which  become  covered  with 
firm  scabs  and  gradually  cicatrize.  The  patient  either  re- 
covers or  suffers  from  relapses  and  the  appearance  of  further 
crops  of  the  lesions.  The  duration  of  the  disease  varies 
from  a  few  weeks  to  several  years.  In  most  cases  the  con- 
stitutional disturbances  occur  only  at  the  period  preceding 
the  development  of  the  eruptions  and  for  a  short  time  after- 
ward. Little  children  frequently  die;  older  children  and 
adults  may  die  of  exhaustion  in  case  extensive  lesions  with 
marked  ulcer ations  develop. 

The  patients  usually  recover  and  pigmented  areas  remain 
for  some  time  where  the  lesions  have  occurred. 

The  disease  appears  to  have  been  known  since  1525,  when 
Oviedo  became  acquainted  with  it  in  St.  Domingo.  It  has 
always  been  very  puzzling  because  it  bears  so  many  resem- 
blances to  syphilis;  but  the  peculiar  raspberry-like  character 
of  the  primary  lesion,  its  disposition  to  occur  upon  the  face, 
mouth,  nose,  eyes,  neck,  limbs,  fingers,  and  toes,  as  well  as 
upon  the  genitals,  seem  to  point  in  another  direction,  and 
all  authorities  now  admit  that  it  is  not  syphilis,  but  an 
independent  disease. 

It  occurs  only  in  tropical  countries,  and  is  most  frequent  in 
equatorial  Africa  on  the  west  coast,  from  Senegambia  to 
Angola.  It  also  occurs  in  West  Soudan,  Algeria,  the  Nile 
Valley,  and  in  the  islands  about  the  east  coast  of  Africa.  It 
has  been  seen  rarely  in  South  Africa.  In  Asia  it  occurs  in 

800 


Morphology — Staining 


801 


Malabar,  Assam,  Ceylon,  Burraah,  Siam,  Malay  Peninsula, 
the  Indian  Archipelago,  Moluccas,  and  China.  It  is  also 
endemic  in  many  of  the  islands  and  archipelagos  of  the 
southern  Pacific. 

The  cause  of  the  disease  was  unknown  until  the  discovery  of 
Treponema  pallidum,  which  opened  a  way  for  its  investiga- 
tion. Castellani*  was  quick  to  seize  the  opportunity,  and  in 
the  same  year  in  which  Schaudinn  and  Hoffmann  discovered 
the  cause  of  syphilis,  announced  a  similar  organism  as  the 
cause  of  yaws.  At  the  time  of  discovery  he  called  it  Spiro- 


Fig.  261. — Yaws  (photograph   by    P.    B.  Cousland,  M.  B.,  Swatow, 

China). 

chaeta  pertenuis  and  Spirochaeta  pallidula,  but  it  is  now  recog- 
nized as  a  treponema  and  is  called  Treponema  pertenue. 

Morphology. — The  organism  so  closely  resembles  Tre- 
ponema pallidum  that  it  is  rather  by  knowing  the  source 
from  which  the  organism  was  derived  than  by  any  morpho- 
logic distinctions  that  the  two  are  separated.  It  measures 
7  to  20  ft  in  length,  is  closely  and  regularly  coiled,  and  is  said 
to  have  rounded  ends. 

Staining. — It  stains  like  its  close  relative,  palely  with 
most  of  the  dyes.  The  silver  nitrate,  the  India  ink  methods, 
and  the  other  methods  of  staining  Treponema  are  all  ap- 

*"Brit.  Med.  Jour.,"  1905,  n,  282,  1280,  1330. 


802  Frambesia  Tropica 

propriate,  both  for  demonstrating  it  in  smears  from  the  le- 
sions or  in  sections  of  tissue. 

Cultivation. — Up  to  the  beginning  of  1912  the  organism 
had  not  yet  been  cultivated. 

Pathogenesis. — Castellani*  has  succeeded  in  infecting 
monkeys  with  the  scrapings  from  yaws  papules.  The  infec- 
tion usually  resulted  in  a  local  lesion,  though  there  was  also 
a  generalized  infection,  for  he  found  treponemata  everywhere 
in  the  lymph-nodes.  When  the  inoculation  material  was 
filtered  and  all  of  the  organisms  removed,  the  infectivity  was 
destroyed.  Blood  and  splenic  substance  from  the  infected 
monkey,  containing  no  organisms  other  than  the  treponemata, 
was  infective  for  other  monkeys.  When  monkeys  success- 
fully inoculated  with  yaws  are  afterward  infected  with  syphil- 
itic virus  they  are  not  immune.  On  the  other  hand,  monkeys 
that  have  successfully  been  inoculated  with  syphilis  are  not 
immune  against  yaws.  Levaditi  and  Nattan-Larrier  f  differ 
from  Castellani  in  this  particular,  and  found  that  monkeys 
infected  with  syphilis  are  refractory  to  yaws.  Castellani 
was  able,  by  means  of  complement-fixation  tests,  to  detect 
different  specific  antibodies  for  syphilis  and  yaws.  Halber- 
stadterj  has  successfully  infected  orang-outangs. 

There  is  no  doubt  but  that  in  their  clinical  manifestations 
arid  in  their  etiology  frambesia  and  syphilis  are  closely 
related. 

*  "Jour,  of  Hygiene,"  1907,  vn,  p.  558. 

t  "Ann.  de  1'Inst.  Pasteur.,"  1908,  xxu,  260. 

t  "Arbeiten  a.  d.  Kaiserl.  Gesund.,"  1907,  xxvi,  48. 


CHAPTER   XXXIII. 
ACTINOMYCOSIS. 

ACTINOMYCES    BOVIS 

General  Characteristics.— A  parasitic,  pathogenic,  aerobic  and 
optionally  anaerobic,  non-motile,  non-flagellate,  non-sporogenous  (?), 
liquefying,  pathogenic,  branched  micro-organism,  belonging  to  the  higher 
bacteria,  staining  by  ordinary  methods  and  by  Gram's  method. 

In  1845  Langenbeck  discovered  that  an  infectious  disease 
of  cattle  known  as  "  wooden  tongue  "  and  "  lumpy  jaw,"  and 
later  as  actinomycosis,  could  be  communicated  to  man. 
The  observation,  however,  was  not  published  until  1878, 


Fig.  262. — Bovine  actinomycosis. 

one  year  after  Bollinger*  had  discovered  the  actinomyces,  the 
specific  cause  of  the  disease. 

Israeli  wrote  the  first  important  paper  upon  actino- 
mycosis as  a  disease  of  man,  though  the  best  paper  on  the 
subject  is  probably  that  by  Bostrom,J  who  made  a  careful 
study  of  the  microscopic  lesions  of  the  disease. 

Its  first  manifestations  are  usually  found  either  about  the 
jaw  or  in  the  tongue,  and  consist  of  considerable  sized  en- 
largements which  are  sometimes  dense  and  fibrous  (wooden 
tongue),  sometimes  suppurative  in  character.  In  sections 
of  tissue  containing  these  nodular  formations,  small  yellow- 

*"  Deutsche  Zeitschrift  fur  Thiermedizin,"  1877. 
t  "Virchow's  Archives,"  1874-78. 
%  "Zeitschrift  fur  Hygiene,"  1889. 
803 


804 


Actinomycosis 


ish  granules  surrounded  by  some  pus  can  usually  be  found. 
These  granules,  when  examined  beneath  the  microscope, 
consist  of  peculiar  rosette-like  bodies — the  "  ray-fungi  "  or 
actinomyces. 

Distribution. — The  actinomyces  is  best  known  as  a 
parasitic  organism  associated  with  actinomycosis.  That  it 
occurs  rather  widely  in  nature  seems  to  be  indicated  by  the 


Fig.  263. — Colony  or  granule  of  actinomyces  in  a  section  through  a 
lesion,  showing  the  Gram-stained  filaments  and  hyaline  material  and 
also  the  pus-cells  surrounding  the  colony  (Wright  and  Brown). 

fact  that  cases  of  infection  have  been  known  to  occur  from  the 
spines  of  barley  and  other  cereals.  Berestnew*  succeeded 
in  isolating  the  organisms  from  hay  and  straw. 

Morphology. — A  complete  ray-fungus  consists  of  several 
distinct  zones  composed  of  different  elements.  The  center 
is  composed  of  a  granular  mass  containing  numerous  bodies 
resembling  micrococci  or  spores.  Extending  from  this  cen- 
ter into  the  neighboring  tissue  is  a  radiating,  branched, 
*  "Centralbl.  f.  Bact.,"  etc.,  Ref.,  1898,  No.  24. 


Morphology  805 

tangled  mass  of  mycelial  threads.  In  an  outer  zone  these 
threads  are  seen  to  terminate  in  conspicuous,  club-shaped, 
radiating  forms  which  give  the  colonies  their  rosette-like 
appearance.  The  clubs  are  inconspicuous  in  the  human 
lesions  of  the  disease. 

The  pleomorphism  of  the  organism  and  the  branched 
network  it  forms  class  it  among  the  higher  bacteria  in  the 
genus  Actinomyces.  When  the  clumps  formed  in  artificial 
cultivations  of  the  parasite  are  properly  crushed,  spread 


Fig.  264. — Actinomyces  granule  crushed  beneath  a  cover-glass, 
showing  radial  striations  in  the  hyaline  masses.  Preparation  not 
stained;  low  magnifying  power  (Wright  and  Brown). 

out,  and  stained,  the  long  mycelial  threads,  0.3-0.5  p.  in 
thickness,  frequently  show  flask-  or  bottle-like  expansions 
— the  clubs — at  the  ends.  These  probably  depend  upon 
gelatinization  of  the  cell-membrane  of  the  degenerating 
parasite.  The  club  is  one  of  the  chief  characteristics  of  the 
organism.  In  sections  of  tissue  the  radiating  filaments  are 
very  distinct,  and  the  terminal  clubs  are  all  directed  outward, 
closely  packed  together,  and  making  the  whole  mass  form 
a  rounded  little  body  often  spoken  of  as  an  "actinomyces 
grain."  When  tissues  are  stained  first  with  carmin  and  then 
by  Gram's  method,  the  fungous  threads  appear  blue-black, 
the  clubs  red.  The  cells  of  the  tissues  affected  and  a  larger 
or  smaller  collection  of  leukocytes  form  the  surrounding 
resisting  tissue-zone. 


806  Actinomycosis 

The  fungus  is  of  sufficient  size  to  be  detected  in  pus, 
etc.,  by  the  naked  eye.  It  can  be  colored,  in  sections  of 
tissue,  by  the  use  of  Gram's  or  Weigert's  stain.  Tissues 
pre-stained  with  carmin,  then  by  Weigert's  method,  show 
beautifully. 

Cultivation. — The  actinomyces  fungus  may  be  grown 
upon  all  the  artificial  culture  media,  as  has  been  fully  shown 
by  Israel,*  Wolff,  and  others. 

To  obtain  a  pure  culture,  material  containing  the  actino- 
myces granules,  secured  so  as  to  be  as  free  as  possible  from 
contaminating  micro-organisms,  is  crushed  between  glass 
plates  or  in  a  mortar,  and  the  crushed  fungi  transferred 
to  plates  or  tubes  as  desired.  The  colonies  appear  as  small 
gray  dots,  and  consist  of  a  translucent,  radiating  filamentous 
network.  If  kept  for  a  few  days  at  37°  C.  they  become 
opaque  and  nodular,  with  radiating  processes  about  the 
periphery.  Still  later  they  develop  a  whitish  downy  ap- 
pearance from  the  formation  of  short  aerial  hyphae.  The 
best  growth  occurs  when  free  access  of  oxygen  is  permitted. 

Blood-serum. — Upon  blood-serum  the  nodular  growths 
present  a  yellowish  or  rust-red  color,  and  are  surrounded 
with  a  whitish  down  of  fine  threads.  The  colonies  adhere 
closely  to  the  culture  media  and  are  so  firm  that  they  crush 
with  difficulty.  If  the  surface  be  scraped,  spores  and  fine 
threads  may  be  secured.  If  the  mass  be  crushed,  branched 
filaments  may  be  secured.  The  colonies  become  confluent 
in  the  course  of  time,  and  a  thick  wrinkled  membrane  is 
produced.  The  growth  liquefies  blood-serum. 

Gelatin. — In  gelatin  puncture  cultures  an  arborescent 
growth  occurs  and  the  gelatin  is  liquefied. 

Agar-agar. — Upon  agar-agar  and  glycerin  agar-agar  the 
growth  is  similar  to  that  upon  blood-serum.  The  agar-agar 
turns  brown  as  the  culture  ages. 

Bouillon. — In  bouillon  the  growth  occurs  in  the  form 
of  large  granules  if  allowed  to  stand  quietly;  of  numerous 
small  granules  if  frequently  shaken  up.  The  granules  are 
similar  in  structure  to  those  formed  upon  the  dense  media. 
The  bouillon  does  not  become  clouded. 

Potato. — Upon  potato  the  growth  resembles  that  upon 
blood-serum,  but  is  slower  in  developing.  The  color  is  red- 
dish-yellow and  the  white  down  early  makes  its  appearance. 

Eggs. — The  organism  can  also  be  grown  in  raw  eggs,  into 
*  "Virchow's  Archives,"  cxv. 


Cultivation 


807 


Fig.  265. — Colony  of  actinomyces  with  well-developed  "clubs"  at 
the  periphery  in  a  nodule  in  the  peritoneal  cavity  of  a  guinea-pig  in- 
oculated with  a  culture  from  another  guinea-pig.  Paraffin  section. 
Low  magnification  (Wright).  (Photograph  by  Mr.  L.  S.  Brown.) 


Fig.  266. — A  colony  of  actinomyces  in  a  nodule  twenty-eight  days 
old  in  the  peritoneal  cavity  of  a  guinea-pig  inoculated  with  a  culture 
from  another  guinea-pig  (Bovine  case).  The  "clubs"  are  well  devel- 
oped and  show  some  indications  of  stratification.  Paraffin  section. 
X  750aprox.  (Wright).  (Photograph  by  Mr.  L.  S.  Brown.) 


8o8 


Actinomycosis 


ABC 

Fig.  267. — Actinomycosis;  glycerin-agar  cultures:  A,  Discrete 
rounded  colonies  after  about  ten  days'  incubation  at  37°  C. ;  B, 
limpet-shaped  colonies  three  and  a  half  months  old  ;  C,  lichen-like 
appearance  frequently  seen  ;  the  growth  is  three  and  a  half  months 
old  (Curtis). 


Pathogenesis  809 

which  it  is  carefully  introduced  through  a  small  opening 
made  under  aseptic  precautions.  In  the  eggs  long, 
branched  mycelial  threads  quite  unlike  the  bacillary  forms 
that  grow  upon  agar-agar  are  formed. 

The  characteristic  rosettes  so  constantly  found  in  the  tis- 
sues are  never  seen  in  artificial  cultures. 

Virulence. — When  the  actinomyces  is  grown  upon  artifi- 
cial media  the  virulence  is  retained  for  a  considerable  time. 

Pathogenesis. — Actinomycosis  is  almost  peculiar  to  bo- 
vine animals,  but  sometimes  occurs  in  hogs,  horses,  and 
other  animals,  and  rarely  in  human  beings.  The  disease 
can  with  difficulty  be  inoculated  into  experiment  animals, 
the  introduced  fungi  either  becoming  absorbed  or  encap- 
sulated by  connective  tissue  and  not  growing.  In  the 
abdominal  cavities  of  rabbits  the  peritoneum,  mesentery,  and 
omentum  show  typical  nodules  containing  the  actinomyces 
rays  in  cases  of  successful  inoculation. 

Mode  of  Infection. — The  manner  by  which  the  organ- 
ism enters  the  body  is  not  positively  known.  In  some  cases 
it  may  be  by  direct  inoculation  with  infectious  pus,  but 
there  is  some  reason  to  believe  that  the  organism  occurs  in 
nature  as  a  saprophyte,  or  as  an  epiphyte  upon  the  hulls 
of  certain  grains,  especially  barley.  Woodhead  has  recorded 
a  case  where  a  primary  mediastinal  actinomycosis  in  the 
human  subject  was  apparently  traced  to  perforation  of  the 
posterior  pharyngeal  wall  by  a  barley  spikelet  accidentally 
swallowed  by  the  patient. 

Cases  of  actinomycosis  are  fortunately  somewhat  rare  in 
human  medicine,  and  do  not  always  occur  in  those  brought  in 
contact  with  the  lower  animals.  The  fungi  may  enter  the  or- 
ganism through  the  mouth  and  pharynx,  through  the  respira- 
tory tract,  through  the  digestive  tract,  or  through  wounds. 

The  invasion  has  been  known  to  take  place  at  the  roots 
of  carious  teeth,  and  is  more  liable  to  occur  in  the  lower 
than  in  the  upper  jaw.  Israel  reported  a  case  in  which 
the  primary  lesion  seemed  to  occur  external  to  the  bone 
of  the  lower  jaw,  as  a  tumor  about  the  size  of  a  cherry, 
with  an  external  opening.  Two  cases  of  the  disease 
observed  by  Murphy,  of  Chicago,  began  with  toothache  and 
swelling  of  the  jaw.  A  few  cases  of  dermal  infection  are 
recorded.  Elsching  *  has  seen  a  case  in  which  calcified 
actinomyces  grains  were  observed  in  the  tear  duct. 
*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  xvm,  p.  7. 


8io  Actinomycosis 

When  inhaled,  the  organisms  enter  the  deeper  portions 
of  the  lung  and  cause  a  suppurative  broncho-pneumonia 
with  adhesive  inflammation  of  the  contiguous  pleura.  After 
the  formation  of  the  pleuritic  adhesions  the  disease  may 


Fig.  268. — Section  of  liver  from  a  case  of  actinomycosis  in  man 
(Crookshank). 

penetrate  the  newly  formed  tissue,  extend  to  the  chest- 
wall,  and  ultimately  form  external  sinuses;  or,  it  may 
penetrate  the  diaphragm  and  invade  the  abdominal  organs, 
causing  interesting  and  characteristic  lesions  in  the  liver 
and  other  large  viscera. 


Lesions  811 

Lesions. — The  degree  of  chemotactic  influence  exerted 
by  the  organism  seems  to  depend  upon  the  tissue  affected, 
upon  the  peculiarity  of  the  animal,  and  upon  the  virulence 
of  the  organism.  When  an  animal  is  but  slightly  suscepti- 
ble, and  especially  when  the  tongue  is  affected,  the  disease 
is  characterized  by  the  formation  of  cicatricial  tissue — 
"wooden  tongue."  If,  on  the  other  hand,  the  animal  be 
highly  susceptible  and  the  jaw-bone  affected,  suppuration, 
with  the  formation  of  abscesses,  osteoporotic  cavities,  and 
sinuses,  are  apt  to  be  noticed.  This  form  of  the  disease  is 
called  "lumpy  jaw"  in  cattle. 

Before  the  nature  of  the  affection  was  understood  it 
was  confounded  with  diseases  of  the  bones,  especially  osteo- 
sarcoma. 

From  the  tissues  primarily  affected  the  disease  spreads 
to  the  lymphatic  glands,  and  eventually  to  the  lungs.  Israel 
has  pointed  out  that  certain  cases  of  human  actinomycosis 
begin  in  the  peribronchial  tissues,  probably  from  inhalation 
of  the  fungi. 

But  few  cases  recover,  the  disease  terminating  in  death 
from  exhaustion  or  from  complicating  pneumonia  or  other 
organic  lesions. 


CHAPTER   XXXIV. 


MYCETOMA,  OR  MADURA-FOOT* 

ACTINOMYCES  MADURA  (VINCENT). 

General  Characteristics.— A  non-motile,  non-flagellate,  sporogen- 
ous  (?),  non-liquefying,  non-aerogenic,  chromogenic,  aerobic  and  option- 
ally anaerobic,  branched,  parasitic  organism  belonging  to  the  higher 
bacteria,  staining  by  ordinary  methods  and  by  Gram's  method,  and 
pathogenic  for  man. 

A  curious  disease  of  not  infrequent  occurrence  in  the 
Indian  province  of  Scinde  and  of  rare  occurrence  in  other 
countries  is  known  as  mycetoma,  Madura-foot,  or  pied  de 
Madura.  Although  described  as  peculiar  to  Scinde,  the  dis- 
ease is  not  limited  to  that  province, 
but  has  been  met  with  in  Madura, 
Hissar,  Bicanir,  Delhi,  Bombay, 
Baratpur,  Morocco,  Algeria,  and  in 
Italy.  In  America  less  than  a 
dozen  cases  of  the  disease  have  been 
placed  on  record.  In  India  it  al- 
most invariably  affects  natives  of 
the  agricultural  class,  and  in  nearly 
all  cases  is  referred  by  the  patient 
to  the  prick  of  a  thorn.  It  usually 
affects  the  foot,  more  rarely  the 
hand,  and  in  one  instance  was  seen 
by  Boyce  to  affect  the  shoulder  and 
hip.  It  is  more  common  in  men 
than  in  women,  individuals  between 

^^  and  forty  years  of  ^  suffer- 
ing  most  frequently,  though  persons 
of  any  age  may  suffer  from  the 

disease.  It  is  insidious  in  onset,  no  symptoms  being  ob- 
served in  what  might  be  called  the  incubation  stage  of  a 
couple  of  weeks'  duration,  except  the  formation  of  a  nodular 
growth  which  gradually  attains  the  size  of  a  marble.  Its  deep 
attachments  are  indistinct  and  diffuse.  The  skin  over  it 
becomes  purplish,  thickened,  indurated,  and  adherent.  The 

812 


Fig.  269. — Madura-foot 
— mycetoma  (Musgrave 
and  Clegg). 


Morphology 


813 


ball  of  the  great  toe  and  the  pads  of  the  fingers  and  toes 
are  the  points  most  frequently  invaded.  The  lesions  pro- 
gress very  slowly,  and  in  the  course  of  a  few  months  form 
distinct  inflammatory  nodes.  After  a  year  or  two  the 
nodes  begin  to  soften,  break  down,  discharge  necrotic  and 
purulent  material,  occasioning  the  formation  of  ulcers  and 
sinuses.  The  matter  discharged  from  the  lesions  at  this 
stage  of  the  disease  is  a  thin  seropus,  and  contains  occasional 
fine  round  pink  or  black  bodies,  similar  to  actinomyces 
"  grains,"  described,  when  pink,  as  resembling  fish-roe; 
when  black,  as  resembling  gunpowder.  It  is  upon  the  de- 
tection of  these  particles  that  the  diagnosis  rests.  Accord- 
ing to  the  color  of  the  bodies  found,  cases  are  divided  into  the 
pale  or  ochroid,  and  melanoid  varieties. 

The  progress  of  the  disease  causes  an  enormous  enlarge- 
ment of    the  affected  part. 
The  malady  is  usually  pain- 
less. 

The  micro-organismal  na- 
ture of  the  disease  was  early  f|r 
suspected.  In  spite  of  the 
confusion  caused  by  some 
who  confounded  the  disease 
with  "guinea- worm,"  Carter 
held  that  it  was  due  to  some 
indigenous  fungus  as  early 
as  1874.  Boyce  and  Sur- 
veyor found  that  the  black 
particles  of  the  melanoid 
variety  consisted  of  a  large 
branching  septate  fungus. 

Pale  Variety.  --  Kan- 
thack  was  the  first  to  prove 
the  identity  of  the  fungus 
with  the  well-known  actino- 
myces, but  there  seems  to  be 
considerable  doubt  about  the 
identity  of  the  species: 

Morphology. — Under  the 
microscope  the  organism  is 
found  by  Vincent*  to  be 

branched  and  belong  to  the  higher  bacteria.     It  consists  of 
*  "Ann.  de  1'Inst.  Pasteur,"  94,  3. 


Fig.  270. — Streptothrix  mad- 
urae  in  a  section  of  diseased  tis- 
sue (Vincent). 


8 14  Mycetoma,  or  Madura-foot 

long,  branched  bacillary  threads  forming  a  tangled  mass. 
In  many  of  the  threads  spores  could  be  made  out.  He 
was  unable  to  communicate  the  disease  to  animals  by  in- 
oculation. 

Cultivation. — Vincent  succeeded  in  isolating  the  specific 
micro-organism  by  puncturing  one  of  the  nodes  with  a 
sterile  pipet,  and  cultivated  it  upon  artificial  media,  acid 
vegetable  infusions  seeming  best  adapted  to  its  growth. 
It  develops  scantily  at  the  room  temperature,  better  at 
37°  C. — in  from  four  to  five  days.  In  twenty  to  thirty 
days  a  colony  attains  the  size  of  a  little  pea. 

Bouillon. — In  bouillon  and  other  liquid  media  the 
organisms  form  little  clumps  resembling  those  of  actino- 
myces.  They  cling  to  the  glass,  thus  remain  near  the 
surface  of  the  medium,  and  develop  a  rose-  or  bright-red 
color.  Those  which  sink  to  the  bottom  form  spheric  balls 
devoid  of  the  color. 

Gelatin. — The  growth  in  gelatin  is  not  very  abundant, 
and  forms  dense,  slightly  reddish,  rounded  clumps.  Some- 
times there  is  no  color.  There  is  no  liquefaction. 

Agar-agar. — Upon  the  surface  of  agar-agar  beautiful 
rounded,  glazed  colonies  are  formed.  They  are  at  first 
colorless,  but  later  become  rose-colored  or  bright  red. 
The  majority  of  the  clusters  remain  isolated,  some  of  them 
attaining  the  size  of  a  small  pea.  They  are  usually 
umbilicated  like  a  variola  pustule,  and  present  a  curious 
appearance  when  the  central  part  is  pale  and  the  periphery 
red.  As  the  colony  ages  the  red  color  is  lost  and  the  colony 
becomes  dull  white  or  downy  from  the  formation  of  aerial 
hyphae.  The  colonies  are  very  adherent  to  the  surface  of  the 
medium,  and  are  almost  of  cartilaginous  consistence. 

Milk. — The  organism  grows  in  milk  without  causing 
coagulation. 

Potato. — Upon  potato  the  growth  of  the  organism  is 
meager  and  slow,  with  very  little  chromogenesis.  The 
color-production  is  more  marked  if  the  potato  be  acid  in 
reaction.  Some  of  the  colonies  upon  agar-agar  and  potato 
have  a  powdery  surface,  either  from  the  formation  of  spores 
or  of  aerial  hyphae. 

Lesions. — Microscopic  study  of  the  diseased  tissues  in 
mycetoma  is  not  without  interest.  The  healthy  tissue  is 
sharply  separated  from  the  diseased  areas,  which  appear 
like  large  degenerated  tubercles,  except  that  they  are  ex- 


Lesions 


815 


tremely  vascular.  The  mycelial  or  filamentous  mass  occu- 
pies the  center  of  an  area  of  degeneration,  where  it  can 
be  beautifully  demonstrated  by  the  use  of  appropriate 
stains,  Gram's  and  Weigert's  methods  being  excellent  for 


Fig.  271. — Melanoid  form  of  mycetoma.  Section  showing  black 
granules  and  general  features  of  the  lesions  as  they  appear  under  a 
low-magnifying  power.  Zeiss  0,1  (James  H.  Wright). 


Fig.  272. — Melanoid  form  of  mycetoma.  showing  structure  and  ap- 
pearance of  the  hyphae  of  the  mycelium  obtained  from  the  granules. 
Zeiss  apochromat;  4  mm.  (James  H.  Wright). 

the  purpose.  The  tissue  surrounding  the  nodes  is  infiltrated 
with  small  round  cells.  Tne  youngest  nodules  consist  of 
granulation-tissue,  whose  development  is  checked  by  early 
coagulation-necrosis.  Giant-cells  are  few. 


8i6 


Mycetoma,  or  Madura-foot 


Not  infrequently  small  hemorrhages  occur  from  the 
ulcers  and  sinuses  of  the  diseased  tissues;  the  hemorrhages 
can  be  explained  by  the  abundance  of  small  blood-vessels 
in  the  diseased  tissue. 


Fig.  273. — Melanoid  form  of  mycetoma.  Two  bouillon  cultures 
showing  the  powder-puff  ball  appearance.  In  one  the  black  granule  is 
seen  in  the  center  of  the  growth  (James  H.  Wright). 


Fig.  274. — Melanoid  form  of  mycetoma.  Potato  culture  of  the 
hyphomycete  obtained  from  the  granules.  The  black  globules  are 
composed  of  a  dark  brown  fluid  (James  H.  Wright). 

The   Melanoid    Form  of  mycetoma  has  been  carefully 
investigated  by  Wright*  and  appears  to  depend  upon  an 
entirely  different  micro-organism  properly  classed  among  the 
*  "Journal  of  Experimental  Medicine,"  vol.  m,  1898,  p.  421. 


The  Melanoid  Form  817 

hyphomycetes.  It  is  probably  identical  with  the  organism 
described  by  Boyce  and  Surveyor. 

In  the  case  studied,  Wright  found  the  diseased  tissues, 
consisting  of  several  of  the  pads  of  the  toes,  to  be  either 
translucent  and  myxomatous  or  yellowish  and  necrotic  in 
appearance.  The  black  granules  were  embedded  in  the 
tissue  and  appeared  mulberry-like  and  less  than  i  mm.  in 
diameter.  They  were  firm,  and  when  enucleated  and 
pressed  between  cover  and  slide  did  not  crush.  Only  after 
digestion  with  a  solution  of  caustic  potash  and  careful 
teasing  could  the  granules  be  resolved  into  the  hyphse  of 
the  mold.  The  central  part  of  the  granule  formed  a  reticu- 
lum,  with  radiating,  somewhat  clavate  elements  projecting 
from  it. 

In  sections  of  tissue  it  was  found  possible  to  stain  the 
fungus  with  Gram's  and  Weigert's  stains,  though  prolonged 
washing  removed  most  of  the  dye. 

Cultural  Characteristics. — Enucleated  granules  carefully 
washed  in  sterile  bouillon  and  then  planted  upon  agar-agar 
afforded  cultures  of  the  mold  in  25  out  of  65  attempts. 

The  growth  began  in  five  or  six  days,  appearing  on  solid 
media  as  a  tuft  of  delicate  whitish  filaments,  springing  from 
the  black  grain,  and  in  a  few  days  covering  the  entire  surface 
of  the  medium  with  a  whitish  or  pale  brown  felt-work. 
Upon  potato  this  felt-work  supports  drops  of  brownish 
fluid.  The  long  branched  hyphae  thus  formed  were  from 
3  to  8  /J.  in  diameter,  with  transverse  septa  in  the  younger 
ones.  The  older  hyphae  were  swollen  at  the  ends.  No  buds 
were  observed.  No  fruit  organs  were  detected.  In  fluid 
media  the  filaments  radiated  from  the  central  grain  with  the 
formation  of  a  kind  of  puff-ball.  Eventually  the  whole 
medium  becomes  filled  with  mycelia  and  a  definite  surface 
growth  forms. 

The  general  characteristics  of  the  fungus  are  well  shown 
in  the  accompanying  illustrations  from  Wright's  paper. 

52 


CHAPTER   XXXV. 
BLASTOMYCOSIS. 

BLASTOMYCES  DERMATITIDIS  (GILCHRIST  AND  STOKES). 

THE  first  case  in  Which  yeasts  or  blastomycetes  were  defi- 
nitely connected  with  disease  seems  to  have  been  published  by 
Busse.*  He  observed  a  case  of  tibial  abscess  in  a  woman 
thirty-one  years  of  age,  who  died  about  a  year  after  coming 
under  observation.  Postmortem  examination  showed  num- 
bers of  broken-down  nodular  formations  upon  the  bones,  and 
in  the  spleen,  kidneys,  and  lungs.  In  all  of  these  lesions  he 
found,  and  from  them  he  cultivated,  an  yeast,  which,  when  in- 
troduced in  pure  culture  into  animals — mice  and  rats — 
proved  infective  for  them.  He  called  the  organism  Sac- 
charomyces  hominis,  and  the  affection  in  which  it  was  found 
"Saccharomycosis  hominis." 

In  May,  1904,  three  months  before  the  appearance  of 
Busse's  paper,  Gilchrist  exhibited  to  the  American  Dermato- 
logical  Association  in  Washington  microscopic  sections  from 
a  case  of  cutaneous  disease  in  which  peculiar  bodies,  recognized 
as  plant  forms,  were  found.  After  the  appearance  of  Busse's 
papers,  Gilchrist  f  more  fully  described  and  illustrated  his 
findings,  calling  the  lesions  "  blastomycetic  dermatitis." 
Though  much  work  upon  pathogenic  blastomycetes  has  been 
published  and  pathogenic  forms  of  these  micro-organisms 
have  been  described  by  Sanfelice,J  by  Rabinowitsch,§  and 
others,  the  chief  and  almost  the  sole  form  in  which  these 
infections  make  their  appearance  is  a  dermal  infection  known 
as  "blastomycetic  dermatitis." 

The  infection  usually  begins  with  the  formation  of  a  papule 
upon  the  face  or  one  of  the  extremities,  which  suppurates  and 
evacuates  minute  quantities  of  viscid  pus.  The  lesion  crusts 

*  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1894,  xvi,  175. 
f  "Johns  Hopkins  Hospital  Reports,"  I,  269,  291. 
t  "Centralbl.  f.  Bakt.  u.  Parasitenk.,"  1895,  xvn,  113,  625;  xvm,  521; 
xx,  219. 

§  "Zeitschrift  fur  Hygiene,"  etc.,  1896,  xxi,  n. 

818 


Blastomycosis  819 

and  begins  to  heal,  but  at  the  periphery  new  and  usually  mi- 
nute foci  of  suppuration  occur,  so  that  while  the  original 
lesion  tends  to  heal  very  slowly,  with  much  cicatricial  for- 
mation, it  is  always  spreading.  The  progress  is  usually 
slow,  and  Gilchrist's  first  case  spread  only  two  inches  in  four 
years. 

Though  the  progress  is  slow,  it  is  sure,  and  there  is  no  tend- 
ency to  spontaneous  recovery  in  most  cases,  nor  is  the  condi- 
tion modified  by  treatment.  The  patients  may  die  from  in- 
tercurrent  disease  or  from  a  generalization  of  the  blastomy- 
cetic  infection,  which  not  infrequently  happens. 

After  the  work  of  Gilchrist  had  made  clear  the  symptoma- 
tology and  parasitology  of  the  disease,  a  number  of  other 


Fig.  275. — Cutaneous  blastomycosis  (Montgomery). 

cases  were  reported,  and  Ricketts*  published  an  excellent 
and  lengthy  summary  of  all  the  cases  with  references  to  all 
of  the  literature  up  to  that  date.  Another  very  interesting 
paper  by  Montgomery ,  f  published  in  1902,  contains  a  splendid 
atlas  of  photographs  of  the  various  lesions  and  of  the  cultures. 
In  addition  to  the  cutaneous  blastomycosis,  a  second  form 
is  also  occasionally  seen,  and  is  known  as  Coccidioidal  granu- 
loma.  It  seems  to  have  been  first  reported  by  Posadas  and 
WernickeJ  and  has  been  carefully  studied  by  Ophiils.§  In 

*  "Jour.  Med.  Research,"  1901,  i,  373. 
t  "Jour.  Amer.  Med.  Assoc.,"  June  7,  1902,  i,  1486. 
J  "Jour,  de  Microorganismen,"  1891,  xv,  14. 

§  "Jour.  Experimental  Medicine,"  1905,  vi,  443.     Ophiils  and  Moffit, 
"Phila,  Med.  Jour.,"  1900,  v,  1471. 


820 


Blastomycosis 


this  form  of  the  disease  the  lesions  are  in  the  internal  organs, 
macroscopically  and  microscopically  resemble  tubercles,  and 
can  only  be  differentiated  from  them  by  the  presence  of  the 
blastomyces  and  the  absence  of  tubercle  bacilli.  The  lungs 
may  be  affected,  and  Walker  and  Montgomery*  mistook  a 
case  for  miliary  tuberculosis  of  the  lungs.  They  also  seem, 
according  to  Evans  f  to  have  a  predilection  for  the  central 
nervous  system. 

There  seems  to  be  little  reason  for  believing  that  there  is 
any  other  difference  than  that  of  distribution  between  the 


Fig.  276. — Giant  cell  from  a  cutaneous  lesion  in  blastomycosis,  showing 
a  group  of  blastomyces  (Montgomery). 

blastomycetic  dermatitis  and  the  blastomycetic  granuloma, 
or  that  they  are  caused  by  different  micro-organisms.  Re- 
garding the  organisms,  however,  we  are  by  no  means  sure 
that  there  are  not  several  species. 

Specific  Organism. — The  organism  presents  a  variety 
of  appearances  which  may  be  thus  brought  together:  First, 
there  are  round  and  elliptical  disk-like  bodies  that  some  regard 
as  spores,  others  as  the  primitive  or  yeast  form.  These 
measure  10  to  30  {*  in  greatest  diameter,  are  distinctly  doubly 
contoured,  highly  refracting,  and,  though  sometimes  clear 

*  "Jour.  Amer.  Med.  Assoc.,"  1902,  xxxvni,  867. 
t  "Jour,  of  Infectious  Diseases,"  1909,  vi,  535. 


Cultivation 


821 


and  transparent,  as  frequently  granular  and  vacuolated. 
From  these  buds  may  grow,  as  in  the  yeasts ;  or  hypha  may 
form,  as  in  oi'dium.  In  artificial  cultivations  the  hypha  may 
form  a  tangled  mycelium. 

Staining. — The  organisms  are  usually  better  found  with- 
out staining.  They  do  not  stain  with  aqueous  anilin  dyes, 
but  are  penetrated  by  warm  thionin,  alkaline  methylene-blue, 
and  polychrome  methylene-blue.  In  sections  of  tissue 
stained  with  hematoxylon  and  eosin  they  show  as  uncolored 


Fig.  277. — Blastomyces  dermatitidis.  Budding  forms  and  mycelial 
growths  from  glucose  agar  (Irons  and  Graham,  in  "  Journal  of  Infectious 
Diseases"). 

circles;  with  thionin  and  alkaline  methylene-blue  they  may 
take  a  blue  color. 

Cultivation. — The  organism  grows  readily  upon  artificial 
media  when  once  started,  but  the  primitive  culture  is  difficult 
to  secure,  because  the  cocci  and  other  associated  organisms 
are  more  numerous  than  the  blastomyces  and  outgrow  it. 
It  seems  most  satisfactory  to  first  infect  a  guinea-pig  with  the 
organism  from  the  skin,  and  then  start  the  cultivation  from 
its  lesions  than  to  attempt  it  directly  from  the  pus  from 
human  dermal  lesions.  When  the  human  lesions  are  internal, 
pure  cultures  are  easily  started. 

Gilchrist  and  Stokes*  were  able  to  start  cultures  directly 

*  "Journal  of  Experimental  Medicine,"  1898,  in,  53. 


822 


Blastomycosis 


from  the  dermal  lesions.  Hiss  and  Zinsser  recommended 
that  this  be  done  by  greatly  diluting  the  culture  material,  so 
as  to  separate  the  contained  organisms  widely. 

Many  culture-media  prove  appropriate,  glycerin  agar-agar 
and  agar-agar  containing  i  per  cent,  of  dextrose  being  ex- 
cellent. When  once  isolated  the  organism  is  easily  kept 
growing  by  transplanting  every  month  or  two. 


Fig.  278. — Cultures  of  Blastomyces  dermatitidis  upon  solid  culture- 
media  (Montgomery). 

The  colonies  appear  in  a  few  days  as  small  round  hemi- 
spheric dots  with  numerous  prickles  about  the  surfaces. 
Later  they  have  a  moldy  appearance  from  the  develop- 
ment of  aerial  hypha.  They  are  almost  purely  aerobic, 
those  on  the  surface  growing  well,  those  deeply  seated  in  the 
medium  scarcely  at  all. 

Agar-agar  Slants. — These  at  first  show  a  creamy  white 
layer  that  becomes  quite  thick,  and  is  moldy  and  fluffy  on  the 
surface.  After  a  few  weeks  the  agar-agar  begins  to  turn 
yellow  and  later  may  become  brown,  though  the  growth  itself 


Lesions  823 

remains  white  and  unchanged.  The  growth  is  firmly  at- 
tached to  the  agar.  When  old,  the  growth  wrinkles. 

Bouillon. — The  growth  is  not  luxuriant.  The  medium 
is  not  clouded  and  contains  fluffy  flocculi  of  stringy  viscid 
material.  Sugars  added  to  the  medium  may  be  fermented. 

Gelatin. — Growth  takes  place  with  aerial  hypha.  Lique- 
faction does  not  occur  or  is  very  slow. 

Potato. — Abundant  growth  with  aerial  hypha. 

Milk. — Not  coagulated,  not  acidified,  slowly  digested. 

There  is  some  difficulty  in  describing  the  cultures,  as  differ- 
ent authors  describe  them  quite  differently,  evidently  having 
different  organisms  or  different  strains  under  observation. 

Pathogenesis. — The  organisms  are  pathogenic  for  guinea- 
pigs,  rabbits,  and  dogs,  in  which  an  abscess,  not  infrequently 
followed  by  a  generalized  infection,  takes  place. 

Lesions. — The  human  lesions  vary  somewhat.  Gilchrist's 
first  case  resembled  lupus  vulgar  is,  other  cases  present  an 
exaggeration  of  the  ulcerative  element.  Cases  have  also 
been  mistaken  for  syphilis.  The  intractable  character  of  the 
lesions  is  suggestive,  and  the  finding  of  the  micro-organisms  in 
the  viscid  pus  is  pathognomonic. 

Upon  section  the  lesions  still  resemble  lupus  and  other  tu- 
berculous lesions,  but  here  again  the  absence  of  tubercle 
bacilli  and  the  presence  of  the  blastomyces  enable  diagnosis 
to  be  made. 

Transmission. — The  disease  is  transmissible.  The  source 
of  infection  is  not  known. 


CHAPTER   XXXVI. 
RINGWORM. 

TRICHOPHYTON  TONSURANS  (MALMSTEN). 

TINKA  trichophytina,  ringworm  of  the  scalp,  herpes  ton- 
surans,  tinea  circinata,  ringworm  of  the  body,  herpes  cir- 
cinatus,  tinea  unguium,  onychomycosis,  tinea  imbricata, 
herpes  desquamans,  tinea  versicolor,  .pityriasis  versicolor, 
erythrasma,  etc.,  are  diseases  with  well-marked  clinical 
manifestations  and  differences,  all  of  which  may  be  compre- 
hended under  the  general  term  dermatomycosis,  and  are 
caused  by  closely  related  forms  of  parasitic  fungi,  whose 
generic  and  specific  differences  are  matters  of  considerable 
confusion. 

That  certain  of  the  diseases  affect  hairy  parts  and  others 
hairless  parts  of  the  body,  that  still  others  occur  about  the 
nails,  and  that  some  are  superficial  and  practically  sapro- 
phytic,  while  others  penetrate  more  deeply  and  are  undoubt- 
edly parasitic,  do  not  necessarily  point  any  more  conclusively 
to  essential  differences  in  the  infecting  organisms  than  to 
accidents  of  infection  and  variations  in  resisting  power.  A 
review  of  the  literature  leaves  the  student  with  a  deplorable 
confusion  of  ideas,  and  a  feeling  that  the  synonomy  is  too 
complicated  and  the  use  of  terms  too  loose  to  permit  of  sys- 
tematic reconstruction. 

The  discovery  of  micro-organisms  in  these  lesions  seems  to 
have  been  made  in  1842  by  Gruby,*  who  found  mycelial 
threads  and  spores  on  and  in  the  hairs,  and  in  1860  by  Hebra,f 
between  the  epithelial  cells.  The  organism  appears  to  have 
been  called  Trichophyton  tonsurans  in  1845  by  Malmsten. 
The  parasitology  of  all  of  the  trichophyton  infections  was 
thoroughly  studied  by  Sabouraud,J  and  the  old  species, 
Trichophyton  tonsurans,  divided  into  eleven  new  species,  to 

*  "Compt.-rendu,"  Paris,  1842,  xv. 

t  "Handbuch  der  spezullen  Path.  u.  Therapie  von  Virchow,"  in,  1860. 

J"Ann.  de  dermat.  et  de  syphilis,"  m,  1892;  iv,  1893;  v,  1894; 
"Monatshefte,"  1896,  576;  "La  Practique  dermatologique.  Tricho- 
phytie,"  1900. 

824 


Morphology 


825 


/* 


which  four  others  have  since  been  added,  so  that  there  are 
now  described,  with  or  without  justification,  Trichophyton 
crateriforme,  T.  acuminatum,  T.  violaceum,  T.  effractum, 
T.  fulmatum,  T.  umbilicatum,  T.  regulare,  T.  pilosum,  T. 
glabrum,  T.  sulphureum,  T.  polygonum,  T.  exsiccatum,  T. 
circonvulatuni,  T.  flavum,  and  T.  plicatili. 

.  In  general  it  is  customary  to  divide  the  organisms  into  two 
groups,  Trichophyton  microsporon  and  T.  megalosporon, 
the  former  having  large,  the  latter  small,  spores. 

Morphology. — The  trichophyton  parasites  form  delicate 
mycelia  composed  of  somewhat  slender  septate  hypha. 
They  can  best  be  observed  by  ex- 
tracting one  of  the  hairs,  including 
its  root,  from  the  diseased  area, 
or  if  the  affection  be  upon  a  hair- 
less part  of  the  body,  by  scraping 
off  some  of  the  epiderm,  and 
mounting  the  material  between  a 
slide  and  cover  in  a  drop  of  caustic 
potash  solution  (20  per  cent.). 
Under  these  circumstances  the 
spores  are  conspicuous  and  so 
numerous  as  to  give  the  impression 
that  they  occur  in  rows  in  a  kind 
of  structureless  zooglea  upon  the 
outside  of  the  hair.  In  some  cases, 
however,  especially  in  Trichophy- 
ton megalosporon,  the  hypha  may 
be  observed  with  the  spores  inside. 
The  hypha  measure  from  2  to  8  ^ 
in  diameter,  are  usually  simple, 
and  rarely  divide.  The  spores  are 
from  2  to  3  ^  in  diameter  in  the 
Trichophyton  microsporon  and  7 
to  8  li  in  T.  megalosporon.  The  former  is  the  more  com- 
mon upon  the  hairless,  the  latter  upon  the  hairy,  portions  of 
the  skin. 

Cultivation. — The  organisms  may  be  secured  in  pure 
culture  without  much  difficulty,  except  for  the  annoying  and 
almost  constant  presence  of  the  associated  bacteria  of  the 
skin.  By  crushing  the  hairs  and  scales  in  a  mortar  with  some 
dilute  KOH  solution,  and  then,  after  thoroughly  distributing 
the  spores  through  the  alkaline  medium  which  dissolves  many 


I 


fl 

!  il 


Fig.  279. — Invasion  of  a 
human  hair  by  trichophy- 
ton :  A,  Points  at  which  the 
parasitic  fungi  coming  from 
the  epidermis  are  elevating 
the  cuticle  of  the  hair  and 
entering  into  its  substance. 
Magnified  200  diameters 
(Sabouraud). 


826 


Ringworm 


of  the  bacteria,  plates  can  be  made  with  high  dilutions,  or 
drops  of  the  fluid  may  be  spread  over  potato,  which  is  an 
excellent  medium  for  the  culture. 

The  culture,  whether  upon  agar-agar,  glycerin  agar-agar, 
glucose  agar-agar,  gelatin,  or  potato,  occurs  in  the  form  of  a 
tuft  of  white  mycelial  filaments  with  aerial  hypha,  looking 
like  a  tiny  white  powder-puff.  Upon  the  surface  of  liquid  cul- 
ture-media the  growth  appears  as  a  thick  wrinkled  pellicle 
with  aerial  hypha  of  velvety  appearance.  As  the  cultures 


Fig.  280. — Trichophyton  tonsurans.     Primary  cultures   twenty  days' 
old  on  maltose  agar-agar.     Natural  size  (Sabouraud). 

grow  older  the  lower  mycelial  growth  becomes  yellowish  and 
wrinkled,  but  the  aerial  hypha  maintain  the  velvety  white  ap- 
pearance. Some  of  the  colonies  are  mammillated,  some  are 
crateriform.  Gelatin  is  liquefied,  the  growth  floating  upon 
the  surface  of  the  fluid.  As  the  cultures  become  very  old  and 
dry  the  velvety  appearance  is  lost  and  the  surface  becomes 
powdery.  The  powder  detaches  only  when  the  growth  is 
touched,  and  does  not  shake  off. 

Pathogenesis. — The    trichophytons    are    pathogenic    for 
man  and  for  the  lower  animals.     They  spread  from  animal  to 


Pathogenesis  827 

animal  by  contact  and  by  inoculation.  Men,  dogs,  cats, 
horses,  sheep,  goats,  and  swine  all  suffer  from  the  infection. 
The  growth  of  the  hypha  between  the  epidermal  layers  causes 
a  chronic  inflammation,  with  hyperemia,  desquamation,  the 
formation  of  some  papules,  and  occasional  pustules.  The 
invasion  of  the  hair-follicles  and  the  growth  of  the  fungi  into 
the  hairs  cause  them  to  become  fragile  and  break  off,  as  well 
as  to  loosen  and  drop  out. 

The  name  "barber's  itch"  results  from  the  frequent  trans- 
mission of  the  infection  by  the  barber's  razors.  The  disease 
is  easily  transmissible  and  precautions  should  always  be 
taken  to  prevent  its  dissemination. 


CHAPTER   XXXVII. 
FAVUS. 

ACHORION    SCHONLEINII   (REMAK). 

FAVUS,  or  tinea  favosa,  is  a  chronic  and  destructive  form  of 
dermatomycosis  occurring  in  man  and  animals,  caused  by  a 
fungus  discovered  in  1839  by  Schonlein,*  and  called  in  his 
honor  Achorion  schonleinii  by  Remak  in  1845.  This  fungus 
is  widely  distributed  and  affects  mice,  cats,  dogs,  rabbits, 
fowls,  and  men.  Among  human  beings  it  usually  occurs  upon 
the  scalp  and  other  hairy  parts  of  the  body,  though  it  may 
also  affect  the  hairless  portions  and  even  attack  the  roots  of 
the  nails.  It  is  more  frequent  in  children  than  in  adults. 
The  fungus  grows  vigorously  and  usually  forms  a  small 
sulphur  yellow  disk  about  the  base  of  a  hair.  The  edges  of 
this  detach,  become  everted,  and  the  whole  eventually  sepa- 
rates, forming  the  "scutulum,"  or  characteristic  lesion  of  the 
disease.  The  reaction  is  more  marked,  the  damage  done 
greater,  and  the  disease  less  tractable  than  in  other  forms  of 
dermatomycosis . 

The  infection  seems  to  take  place  in  most  cases  by  way  of 
the  hair-follicles,  and  the  mycelia  of  the  fungi  grow  into  and 
about  the  hairs,  invading  the  epiderm,  and  causing  atrophy 
of  the  hair-follicles  by  pressure.  Beneath  and  around  the 
scutulum,  which  consists  chiefly  of  the  fungi,  an  inflamma- 
tory reaction  takes  place,  and  leukocytic  invasion  and  ulcer- 
ation  cause  the  scutulum  to  separate. 

Although  usually  confined  to  the  skin ,  the  favus  infection 
may  extend  to  the  mucous  membranes,  and  Kaposi  and 
Kundratf  have  reported  a  case  in  which  favus  fungi  were 
found  to  have  invaded  the  stomach  and  intestines. 

The  disease  runs  a  course  sometimes  extending  over  many 
years.  Crocker  J  mentions  a  case  that  recovered  after  thirteen 
years.  It  may  remain  localized  upon  the  scalp  or  may  spread 

*  Muller's  "Archiv.,"  1839. 
f  "Ann.  de  Dermat.  et  de  Syph.,"  1895,  p.  104. 
J  "Diseases  of  the  Skin,"  Phila.,  1903,  p.  1276. 
828 


The  Specific  Organism 


829 


itself  over  much  of  the  skin  surface.  When  the  lesions  are 
large  they  give  off  an  odor  suggesting  that  peculiar  to  white 
mice.  In  recovering,  the  lesions  leave  considerable  cicatricial 


,   - 


Fig.  281 . — Favus.  Hairs  of  a  child  infected  with  Achorion  schonleinii. 
A,  Magnified  260  diameters;  B,  75  diameters.  The  large  rounded  bodies 
are  droplets  of  air  which  always  appear  after  treatment  with  40  per  cent, 
potash  solution.  The  linear  threads  are  the  fungi.  Some  are  without 
spores,  others  contain  rows  of  spores  (Sabouraud). 

scarring,  and  atrophy  of  hair-follicles,  sweat,  and  sebaceous 
glands  is  inevitable. 

The  Specific  Organism. — The  Achorion  schonleinii  is 
probably  better  regarded  as  a  group  of  closely  related  organ- 


830  Favus 

isms  than  as  a  single  one.  Indeed,  Quincke  has  described 
three  species,  though  they  are  not  yet  generally  accepted. 

The  organism  can  be  studied  by  extracting  a  hair  and  exam- 
ining it  in  KOH  or  NaOH  solution  (20  per  cent.),  or  by 
teasing  a  scutulum  in  the  same  medium  and  examining  with 
a  low  power.  Sections  of  the  skin  may  also  be  made  when 
possible. 

The  fungus  resolves  itself  into  mycelial  threads  and  spores. 
The  scutulum  consists  of  masses  of  spores  at  the  center  and 
about  the  hair,  with  mycelia  containing  spores  at  the  edges. 


Fig.  282. — Achorion schonleinii.  Fig.  283. — Achorion  schonleinii. 

Four  weeks'  old  culture  upon  beer-  Pure  culture,  four  weeks  old,  on 

wort  agar-agar  (Kolle  and  Wasser-  beerwort     agar-agar    (Kolle    and 

mann).  Wassermann). 

From  the  mycelium  hypha  are  given  off,  the  ends  being 
knobbed  or  clavate. 

The  mycelial  threads  are  highly  refractile,  contain  granular 
protoplasm,  and  are  of  varying  thickness.  Sometimes  the 
terminal  hypha  are  simple,  sometimes  they  fork,  the  ends  are 
.always  clavate.  The  hypha  give  off  buds  at  right  angles 
along  their  course. 

The  spores  are  oval,  doubly  contoured,  as  a  rule,  but  may  be 
round  or  pointed  and  more  or  less  polyhedral.  They  measure 
3  to  8  ft  in  length  and  3  to  4  f*  in  breadth.  They  form  the 
great  central  mass  of  the  scutulum,  which  is  the  oldest  part. 
Together  with  them  one  finds  a  number  of  detritus  granules, 
fat-droplets,  and  occasional  swollen  epidermal  cells. 

Cultivation. — The  cultivation  of  the  achorion  is  quite 
easy  if  care  be  used,  for  the  central  part  qf  each  scutulum 
contains  pure  cultures  of  the  organism.  The  best  method  is 


Pathogenesis  831 

probably  that  of  Krai,*  which  is  as  follows:  "A  good  deal  of 
the  material  from  the  scutula  is  rubbed  up  in  a  porcelain 
mortar  dish  with  previously  heated  diatomaceous  earth,  with 
a  porcelain  pestle,  without  exerting  too  much  pressure. 
Melted  agar-agar  tubes  are  then  inoculated  with  two  or  three 
loopfuls  of  the  crushed  material  and  poured  into  Petri  dishes. 
Greater  dilution  can  be  made  if  desired.  The  plates  are  ex- 
amined after  forty-eight  hours. 

Cultures  may,  however,  be  directly  made  with  material 
from  the  center  of  a  scutulum.  Agar-agar  should  be  used,  as 
the  cultures  grow  best  at  the  body  temperature.  The 
young  colonies  that  appear  in  forty -eight  hours  can  easily  be 
transplanted  by  fishing  under  a  lens. 

The  best  medium  was  found  by  Sabouraud  to  consist  of 
maltose,  4;  peptone,  2;  fucus  crispi,  1.5;  water,  100. 

As  the  colonies  eventually  become  quite  large  it  is  recom- 
mended that,  instead  of  tubes,  they  be  made  in  Erlenmeyer 
flasks,  the  transplanted  little  colonies  being  placed  at  the 
center  of  the  medium  congealed  upon  the  bottom  of  the  flask. 

The  appearance  of  the  cultures  varies  considerably. 
Plaut  gives  two  principal  varieties :  ( i )  The  waxy  type — a  yel- 
lowish mass  of  a  waxy  character  with  radiating  folds  and  a 
central  elevation.  As  a  rule  no  aerial  hypha,  but  occasionally 
short  aerial  hypha. 

(2)  The  downy  type — this  forms  a  white  disk  with  a  velvety 
or  plush-like  covering  of  white  aerial  hypha.  Sometimes  in- 
stead of  white  the  color  is  yellowish  or  reddish.  A  marked 
dimple  with  a  smaller  elevation  usually  occurs  in  the  middle, 
and  there  may  be  radial  folds. 

Pathogenesis. — The  micro-organism  is  pathogenic  for 
mice,  rabbits,  cats,  dogs,  hens,  and  men,  in  all  of  whom  typical 
scutula  form.  Scutulum  formation  has  not  been  observed  in 
guinea-pigs.  The  disease  readily  spreads  from  animal  to 
animal  by  direct  contact,  and  by  indirect  contact  by  the  use 
of  combs,  hair-brushes,  and  similar  objects.  On  account  of 
its  chronicity,  its  obstinacy,  its  disfigurement,  and  its  trans- 
missibility  it  is  a  dangerous  disease,  and  one  that  requires 
prompt  isolation  of  the  patient  and  the  utmost  care  for  the 
prevention  of  contagion. 

*See  Plaut,  in  Kolle  and  Wassermann's  "Pathogene  Mikroorgan- 
ismen,"  i,  p.  608. 


BIBLIOGRAPHIC  INDEX 


ABBOTT,  101,  102,  187,  188,  238, 
250,  365,  454.  460,  628,  652 

Abbott  and  Bergey,  629,  631 

Abbott  and  Gildersleeve,  452,  759 

Abel,  130,  5*11,  588,  672 

Abel  and  Claussen,  611 

Abel  and  Loffler,  638,  648 

Abelous,  368 

Achard,  665 

Adami,  78,  86,  87 

Adami  and  Chapin,  656 

Afanassiew,  488 

Agramonte,  576 

Agramonte,  Reed,  and  Carroll,  577 

Agramonte,  Reed,  Carroll,  and 
Lazear,  576 

Alav,  485 

Albrech  and  Ghon,  428 

Alessi,  101 

Alt,  449 

Altmann,  269 

Alvarez  and  Tavel,  757 

Anaximander,  17 

Anderson  and  Rosenau,  123,  161 

Andrewes  and  Gordon,  341 

Andrews,  211 

Anjeszky,  188 

Aoyama,  585 

Aristotle,  17 

Arloing,  114,  409,  745,  75O 

Arnaud,  688 

Arning,  771 

Arnold,  205 

Arrhenius,  26 

Arustamoff  and  Vignal,  42 

Aschoff,  131 

Aschoff  and  Gaylord,  200 

Atkinson,  473 

Audanard,  368 

Auld,  498 

Axenfeld,  424,  448,  501 


BABES,  355,  368,  419,  474,  478,  479, 

520,  784,  781 

Babes  and  Cornil,  478,  608 
Babes  and  Ernst,  34,  175 
Babes  and  Lepp,  117,  422 
53 


Babes  and  Proca,  747 

Bail,  146,  150 

Baker,  557 

Baldwin,  384 

Baldwin  and  Stewart,  383 

Baldwin  and  Trudeau,  742,  744 

Banti,  256,  501 

Barbagallo  and  Casagrandi,  707 

Barker,  368 

Barron,  555 

Bass,  539 

Bassett  and  Duval,  700 

Bassett-Smith,  334,  335,  522,  523 

Batement,  562 

Batzaroff,  594 

Baumgarten,  89,  477,  729,  731,  732, 

782,  783 

Baumgarten  and  Walz,  745 
Bauzhaf  and  Steinhardt,  160 
Beattie  and  Dickinson,  568 
Beck  and  Pfeiffer,  517 
Beck  and  Proskauer,  59,  726 
Becker,  348 
Beckman,  657 

Beebe,  Biggs,  and  Park,  468 
Behring,   26,    116,    138,    155,    157, 

214,  392,  471,  506,  729,  737,  747, 

753 

Behring  and  Kitasato,  117,  160 
Behring  and  Nissen,  129 
Behring  and  Nocht,  213 
Belfanti  and  Carbone,  140,  163 
Beninde,  728 
Bensaude,  665 
Benzancon,  443 
Berestneff,  43 
Berestnew,  804 
Berg,  484 

Bergell  and  Meyer,  649 
Bergey,  629 

Bergey  and  Abbott,  629,  631 
Bergholm,  85 
Berkefeld,  208 
Bernheim,  746 

Bernheim  and  Hildebrand,  82 
Bertarelli,  790 
Bertarelli  and  Bocchia,  211 
Bertarelli  and  Volpino,  790 
833 


834 


Bibliographic  Index 


Bertrand  and  Phisalix,  117,  118,161 

Besredka,  357,  424,  490,  638 

Besredka  and  Steinhardt,  124 

Bettencourt  and  Franca,  429 

Beyer,  Rosenau,  Parker,  and  Fran- 
cis, 580 

Beyerinck,  76 

Bezancon,  364 

Bielonovsky,  595 

Biggs,  467 

Biggs,  Park,  and  Beebe,  468 

Bignami,  525 

Billroth,  24,  282 

Biondi,  82,  199 

Biondi  and  Heidenhain,  199 

Birch-Hirschfeld,  727 

Birt  and  Lamb,  522 

Bitter  and  Sternberg,  70 

Bittu  and  Klemperer,  757 

Blanchard,  27,  537,  544 

Blase  and  Russo-Travali,  464 

Blum,  368 

Blumer,  368 

Boas-Oppler,  83 

Bocchia  and  Bertarelli,  2 1 1 

Bockhart,  86,  348 

Boehm,  23 

Boland,  367 

Bellinger,  803 

Bolton,  130,  1 68 

Bolton  and  Globig,  237 

Bolton  and  McBryde,  679 

Bolton  and  Pease,  62 

Bomstein,  468 

Bonhoff,  631 

Bonney  and  Foulerton,  501 

Bonome,  116,  781 

Bonome  and  Gros,  63 

Bonome  and  Viola,  62 

Bordet,  26,  120,  140,  141,  144,  146, 
163,  166,  170,  322,  787 

Bordet  and  Gay,  167 

Bordet  and  Gengou,  119,  320,  335, 
488,  489,  490,  491 

Bordoni-Uffreduzzi,  372,  497,  501 
765 

Borrel,  595 

Borrel  and  Roux,  391,  398 

Bostrom,  803 

Botkin,  261,  262,  263 

Bowhill,  589 

Boxmeyer,  McClintock,  and  Siffer, 
68 1 

Boyce,  812 

Boyce  and  Surveyor,  813,  817 

Brault,  555 

Braun,  705,  708 

Brebeck-Fischer,  485 

Brefeld,  46,  48 


Breinl,  550 

Brieger,  391,  612,  638 

Brieger  and  Cohn,  391 

Brieger  and  Ehrlich,  128,  376 

Brieger  and  Frankel,  91,  391,  406, 

461,  612,  638 
Brown,  440,  441,  443,  444,  455,  572, 

807 

Brown  and  Wright,  804,  805 
Bruce,  520,  522,  560,  562 
Bruce  and  Nabarro,  557,  561 
Bruce,  Nabarro,  and  Greig,  561 
Bruck  and  Neisser,  798 
Bruck  and  Wassermann,  797 
Bruck,  Wassermann,  and  Neisser, 

318,  319,  320,  322 
Bruckner  and  Galasesco,  793 
Brumpt,  557, 561, 562,  706,  707,  708 
Brunner,  686 
Buchner,  128,  129,  165,  263,  265, 

622 

Buchner  and  Daremberg,  141 
Buchner  and  Nuttall,  128 
Buerger,  495,  496,  504 
Bujwid,  159 

Bullock  and  Hunter,  367 
Bumm,  348,  431,  462 
Burri,  792 

Burroughs  and  McCollum,  472 
Busse,  818 

Buswell  and  Kraus,  648 
Biitschli,  525 
Buxton,  663 

Buxtori  and  Coleman,  66 1 
Buxton  and  Torry,  126 
Buxton  and  Vaughan,  151 


CABOT,  419 

Cadio,  Gilbert,  and  Roger,  754 

Calkin,  30 

Calmette,  117,  161,  162,  368,  595, 
650,  741 

Calmette  and  Guerin,  753 

Cameron,  732 

Camus  and  Gley,  163 

Canon,  514 

Canon  and  Pfeiffer,  26 

Cantani,  612 

Capaldi,  659 

Carbone  and  Belfonti,  140 

Cardan,  18 

Carmona  y  Valle,  576 

Carrasquilla,  774 

Carroll,  263,  576,  578 

Carroll  and  Reed,  576,  580 

Carroll,  Reed,  and  Agramonte,  577 

Carroll,  Reed,  Lazear,  and  Agra- 
monte, 576 


Bibliographic  Index 


835 


Carter,  576,  577,  813 
Carteras  and  Hughes,  506 
Casagrandi  and  Barbagallo,  707 
Castellani,  26,  557,  800,  80 1,  802 
Catanni  (Jr.),  518 
Celli,  525,  688,  699 
Celli  and  Fiocca,  690 
Celli  and  Marchiafava,  423 
Celli-Shiga,  664 
Centanni  and  Tizzoni,  746 
Chagas,  564,  565 
Chamberland,  208,  409,  410 
Chamberland  and  Roux,  116,  377, 

412 
Chantemesse,  633,  644,  648,  650, 

651,  760 
Chantemesse  and  Widal,  647,  648, 

699 

Chapin  and  Adami,  656 
Charrin,  116,  365 
Charrin  and  d'Arsonval,  62 
Charrin  and  Roger,  102,  149,  760 
Chauffard  and  Quenu,  398 
Chauveau,  116,  409 
Cheinisse,  442 
Chenot  and  Picq,  784 
Chester,  32,  41,  278,  279 
Chevreul  and  Pasteur,  21 
Cheyne,  98 
Christmas,  435,  437 
Christy,  Button,  and  Todd,  557 
Cienkowsky,  690 
Citron,  334 

Citron  and  Wassermann,  328 
Ciuffo,  798 
Clark,  476 

Clarke  and  Miller,  211 
Claudius,  211 
Claussen  and  Abel,  611 
Clegg,  767,  770 
Clegg  and  Musgrave,  692,  694,  695, 

696,  812 

Cobbett,  1 06,  130,  475 
Cohn,  36,  334,  448 
Cohn  and  Brieger,  391 
Cohnheim,  711 
Colbach,  23 

Coleman  and  Buxton,  66 1 
Coley,  63,  358 
Colla,  103 

Comte  and  Nicolle,  546,  571 
Comus  and  Gley,  169 
Conn,  95 
Conradi,  406 

Conradi  and  Drigalski,  656 
Cooley  and  Gelston,  90 
Cooley  and  Vaughan,  669 
Coplin,  181,  715 
Cornet,  711 


Cornevin,  114,  377 
Cornil  and  Babes,  478,  608 
Councilman,  355,  424,  476 
Councilman  and  Lafleur,  27,  688, 

689,  690,  691 
Councilman,  Mallory,  and  Pearce, 

467 

Countess  del  Cinchon,  525 
Courmont,  745 
Cousland,  80 1 
Cowie,  757 
Craig,  692,  693,  696 
Crandenigo,  380 
Creite,  163 
Crocker,  828 
Crocq,  420 
Crooke,  355 
Crookshank,  810 
Cruveilhier,  160 
Cumston,  671 
Cunningham,  573 
Curry,  511 
Curtis,  44,  389,  4°4.  406,  509,  724, 

808 

Gushing,  640,  643,  664,  665 
Czaplewski,  757,  764,  765 
Czaplewski  and  Hensel,  488 
Czenzynke,  514 
Czerny,  358 


D ALTON  and  Eyre,  520 

Daniels,  196 

Danysz,  686 

Daremberg  and  Buchner,  141 

Darling,  573,  574,  575 

d'Arsonval  and  Charin,  62 

Davaine,  24,  401 

Davaine  and  Pollender,  25 

Davidson,  524 

Davis,  443,  444,  445,  488,  51? 

Dean,  640 

De  Foe,  581 

Deichler,  488 

Delage,  30 

Delepine,  214 

Delezene,  121,  165 

Delius  and  Kolle,  518 

Denecke,  622,  623,  624,  631 

de  Mondeville,  22 

Denny,  452,  455,  468 

Denys,  742 

Denys  and  van  de  Velde,  349 

De  Renzi,  506 

De  Schweinitz,  68 1,  736,  747 

De  Schweinitz  and  Dorset,  678 

De  Schweinitz  and  Veasy,  448,  449 

Descos  and  Nicholas,  87 

de  Silvestri,  687 


836 


Bibliographic  Index 


Detre,  322 

Detweiler,  90 

Deutsch,  115 

Deutsch  and  Feistmantel,  115 

Devell,  591 

Deycke,  237,  688 

Dickinson  and  Beattie,  568 

Dineur,  150 

di  Vestea  and  Maffucci,  747 

Dobbin,  383 

Doderlein,  486 

Doderlein  and  Winterintz,  85 

Doflein,  372,  551 

Donitz,  132,  391,  398 

Donne,  799 

Donovan,  566 

Donovan  and  Leishman,  27 

Dopter  and  Vaillard,  702 

Dorset,  679,  722,  723 

Dorset  and  De  Schweinitz,  678 

Dorset,  McBryde,  and  Niles,  679 

Douglas  and  Wright,  127,  307,  316 

Doutrelepont     and     Matterstock, 

757 

Draper,  670 
Dreyfus,  670 

Drigalski  and  Conradi,  656 
Droba,  640 
Drysdale,  560 
Dubarre  and  Terre,  756 
du  Bary,  49 
Dubois,  454 
Du  Bois-Reymond,  62 
Duboscq  and  Leger,  707 
Ducrey,  88,  442,  443,  766 
Dujardin  and  Ehrenberg,  29 
Dunbar,  631 
Dunham,  240,  241,  379,  383,  458, 

662,  669,  673 
Dunham  and  Park,  699 
Durham,  140,  665 
Durham  and  Gruber,  149 
Durme,  347 

Dutton,  547,  554,  555,  556,  557 
Dutton  and  Forde,  27,  557,  562, 

563 

Dutton  and  Todd,  547,  551,  557 
Dutton,  Todd,  and  Christy,  557 
Duval,  763,  767,  768,  770 
Duval  and  Bassett,  700 
Duval  and  Vedder,  700 


EAGER,  582 

Eberth,  26,  293,  368,  632,  644,  760 

Effront,  68 

Ehlers,  368 

Ehrenberg,  19,  292,  293 

Ehrenberg  and  Dujardin,  29 


Ehrlich,  26,  109,  117,  118,  131,  132, 
136,  138,  143,  144,  145,  150,  153, 
157,  158,  159,  162,  170,  182,  183, 
185,  186,  187,  190,  391,  461,  471, 
713,  7i5,  719,  764 

Ehrlich  and  Brieger,  128,  376 

Ehrlich  and  Morgenroth,  120,  122, 
131,  163,  166,  322 

Eichhorn  and  Mohler,  779,  783,  784 

Eisenberg,  150,  278,  292,  293 

Eisner,  650 

Elders,  478 

Ellermann,  479 

Elmassian  and  Morax,  467 

Elsching,  809 

Elser,  426 

Elser  and  Huntoon,  430 

Eisner,  652,  653,  656,  673 

Emery,  453 

Emmerich,  666 

Emmerich  and  Low,  77,  367 

Emory,  83 

Empedocles,  17 

Endo,  659 

Engle  and  Reichel,  421 

Eppinger,  43 

Ernst,  159,  365,  368,  383,  384 

Ernst  and  Babes,  34,  175 

Ernst  and  Robey,  150 

Escherich,  37,  293,  664,  666 

Esmarch,  223,  237,  246,  250,  251, 
261,  288 

Evans,  219,  820 

Evans  and  Russell,  218 

Eyre,  50 

Eyre  and  Dalton,  520 


FARRAN,  387 

Fasching,  507 

Favre,  798 

Fehleisen,  350,  361 

Fehling,  240,  737 

Feistmantel  and  Deutsch,  115 

Feletti  and  Grassi,  534 

Fermi,  71 

Fermi  and  Pernoss,  391 

Fermi  and  Salsano,  756 

Ferran,  617 

Fick,  477 

Ficker,  152 

Field,  390 

Finkelstein,  368 

Finkler  and  Prior,  619,  620,  621, 

622,  623,  631 
Finlay,  576,  577 
Fiocca,  189,  688,  699 
Fiocca  and  Celli,  690 
Firth,  572,  573 


Bibliographic  Index 


837 


Fisch,  747 

Fischel  and  Wunschheim,  1 30 

Fischer,  135 

Fish,  147 

Fitzpatrick,  596 

Flatten,  424 

Flexner,  43,  355,  373,  378,  380,  424, 

427,  428,  429,  430,  467,  689,  699, 

702,  704,  791 
Flexner  and  Harris,  644 
Flexner  and  Jobling,  430 
Flexner  and  Noguchi,  162,  163,  390 
Flexner  and  Welch,  377,  383,  464 
Flournoy,     Norris,     and     Pappen- 

heimer,  550 
Fliigge,  64,  94,  127,  129,  181,  216, 

217,  292,  293,  294,  295,  365,  370, 

385,  386,  424,  493,  601,  602,  728 
Foa,  501 
Fodor,  127 
Forde,  555,  556 
Forde  and  Button,  27,  557,  562, 

563 

Forneaca,  364 
Forssner,  146 
Foulerton,  42 

Foulerton  and  Bonney,  501 
Fournier  and  Gilbert,  677 
Fox  and  Longcope,  493 
Fracastorius,  22 
Franca  and  Bettencourt,  429 
Frances,     Beyer,     Rosenau,     and 

Parker,  580 
Francis  and  Grubs,  73 
Franke,  477 
Frankel,  64,  106,  261,  262,  273,  294, 

295,  355,  377,  407,  4ii,  424,  425, 
477,  491,  492,  501,  508,  606,  607, 
611,  612,  614,  622,  626,  634,  644 

Frankel  and  Brieger,  91,  391,  406, 
461,  612,  638 

Frankel  and  Pfeiffer,  346,  351,  386, 
387,  388,  400,  402,  403,  459,  503, 
599,  607,  621,  626,  711,  725,  776 

Frankel  and  Weichselbaum,  88, 
373,  492,  509 

Frankel  and  Wollstein,  491 

Frankforter,  219 

Frankland,  292,  293 

Fredericq,  127 

Freire,  576 

Freymuth,  618 

Friedlander,  85,  183,  450,  507,  508, 
509,  510,  511,  512,  785,  786 

Frisch,  368 

Frosch,  463 

Frosch  and  Kolle,  357 

Frost,  64,  251,  252,  254,  255,  288, 
290 


Frothingham,  421 

Frugoni,  725 

Fuller,  225,  291 

Fulleborn,  552 

Fulleborn,  Mayer,  and  Martin,  548 

Funck  and  Metschnikoff,  121 

GABBET,  716,  764,  772 
Gabbi,  501 

Gaffky,  361,  409,  632,  644,  689 
Gaffky  and  Koch,  688 
Galasesco  and  Bruckner,  793 
Galeotti,  72 

Galli-Valerio,  63,  592,  688 
Gamaleia,  493,  499,  612,  625,  628, 

631 

Garini,  240 
Garre,  86,  348 
Gartner,  660,  664,  665,  675 
Gaspard,  21 

Gauss  and  Schumburg,  35 
Gauthier,  798 
Gay,  123 

Gay  and  Bordet,  167 
Gay  and  Southard,  123,  124 
Gaylord  and  Aschoff,  200 
Geddings  and  Wasdin,  577 
Gelston,  90 

Gelston  and  Cooley,  90 
Gelston  and  Marshall,  90 
Gengou,  170 
Gengou  and  Bordet,  119,  320,  335, 

488,  489,  490,  491 
Germano  and  Maurea,  645 
Gessard,  72,  292,  364 
Gheorghiewski,  119,  367 
Ghon,  589 

Ghon  and  Albrech,  428 
Ghoreyeb,  789 
Ghriskey,  340,  341 
Ghriskey  and  Robb,  81 
Gibier,  102 
Gibson,  159 
Giemsa,    197,   480,   550,   706,   788, 

790,  79i,  797 
Gilbert  and  Fournier,  677 
Gilbert,  Cadio,  and  Roger,  754 
Gilchrist,  44,  818,  819,  823. 
Gilchrist  and  Stokes,  818,  821 
Gildersleeve  and  Abbott,  452,  759 
Gilliland  and  Pearson,  753 
Gley  and  Camus,  163,  169 
Globig  and  Bolton,  237 
Goldhorn,  788 
Goldschmidt,  427 
Golgi,  525,  527 
Goodby,  82 
Goodsir,  282 
Goodwin  and  Sholly,  430 


838 


Bibliographic  Index 


Goppert,  424 

Gorden,  192 

Gordon,  584,  615 

Gordon  and  Andrewes,  341 

Gorgas,  580 

Gorham,  73,  452 

Gottschalk  and  Immerwahr,  85 

Gottstein,  122 

Gourvitsch,  707 

Graham,  383,  384 

Graham  and  Irons,  44,  821 

Gram,  182,  183,  184,  185,  198,  343, 
350,  35i,  360,  361,  362,  364,  365, 
369,  374.  377,  379,  385,  386,  399, 
400,  403,  423,  425,  426,  430,  431, 
432,  433,  437,  439,  442,  443,  446, 
447,  449,  450,  451,  453,  480,  489, 
492,  494,  495,  507,  509,  514,  520, 
548,  550,  581,  583,  584,  598,  601, 
602,  604,  608,  619,  625,  631,  632, 
633,  675,  667,  676,  677,  678,  679, 
682,  684,  699,  710,  714,  719,  760, 
762,  764,  775,  776,  785,  787,  803, 
805,  806,  812,  815,  817 

Gram  and  Weigert,  185 

Grassi,  529 

Grass!  and  Feletti,  534 

Grawitz,  45,  485,  486 

Gregorieff,  687 

Greig,  Bruce,  and  Nabarro,  561 

Griffon  and  Le  Sours,  443 

Grigorjeff  and  Ukke,  377 

Grimme,  175 

Grixoni,  387 

Grohmans,   127 

Gromakowsky,  359 

Gros  and  Bonome,  63 

Grosset,  486 

Gruber,  140,  260 

Gruber  and  Durham,  149 

Gruber  and  Wiener,  616 

Griibler,  175,  187,  653,  788 

Grubs  and  Francis,  73 

Gruby,  484,  824 

Griinbaum,  645 

Griinbaum  and  Widal,  649 

Grysez  and  van  Steenberghe,  87 

Gscheidel  and  Traube,  127,  166 

Guarniere,  497 

Guerin  and  Calmette,  753 

Guidi,  485 

Guiteras,  580 

Gunther,  231,  283,  289,  295,  344, 
376,  631 

Gwyn,  643,  665 

HAECKEL,  29 

Haffkine,  114,  585,  586,  596,  597, 
616,  617,  646 


Halberstadter,  802 

Hall,  569 

Hallein,  485 

Hallier,  282 

Hamburger,  651 

Hamerton,  562 

Hamilton,  476 

Hankin,  64,  102,  128,  410 

Hankin  and  Leumann,  588 

Hankin  and  Wesbrook,  406 

Hansen,  26,  68,  486,  762,  763,  765 

Harris,  698 

Harris  and  Flexner,  644 

Harrison,  562 

Harvey,  18 

Hashimoto,  612 

Hasslauer,  85 

Haupt,  450 

Hauser,  369,  370 

Havelburg,  576,  594 

Hebra,  824 

Hegar,  85 

Heidenhain,  199 

Heidenhain  and  Biondi,  199 

Heider,  631 

Heim,  345,  363,  484,  515,  635,  609, 

667 

Heiman,  434 
Hektoen,  732 
Henle,  23 

Hensel  and  Czaplewski,  488 
Herman,  98 
Herzog,  593 
Hesse,  265,  284,  658 
Hesse  and  Liborius,  265 
Hewlett,  132,  136,  665 
Hewlett  and  Nolen,  469 
Heyman-Sticher,  728 
Heymans,  132 
Hildebrand,  118 
Hildebrand  and  Bernheim,  82 
Hill,  174,  250,  304,  657 
Hippocrates,  687 
Hirsch,  354 
Hiss,  50,  216,  353,  427,  494,  495, 

498,  505,  636,  654,  655,  656,  673, 

702,  741,  792 
Hiss  and  Russell,  699 
Hiss  and  Zinsser,  41,  225,  352,  362, 

424,  496,  497,  506,  632,  663,  777, 

822 

Hochst,  657,  744 
Hodenpyl,  731 
Hodenpyl  and  Prudden,  749 
Hoffa,  405 

Hoffmann  and  Prowazek,  799 
Hoffmann  and  Schaudinn,  26,  82, 

787,  788,  799,  801 
Hofmann,  468,  474,  547,  636,  793 


Bibliographic  Index 


839 


Hogyes,  414,  419 

Holmes,  23 

Hoist,  95,  356 

Horder,  517 

Howard,  463,  474,  501,  512,  541 

Howard  and  Perkins,  359,  360 

Howard,  Jr.,  384 

Hiickel  and  Losch,  48 

Hughes,  522 

Hughes  and  Carteras,  506 

Humer,  640 

Hunter  and  Bullock,  367 

Huntoon  and  Elser,  430 

Hunziker,  260 

Hiippe,  293,  410,  60 1,  607,  608 

Hiippe  and  Wood,  411 

Huxley,  30 


IMMERWAHR  and  Gottschalk,  85 

Inchley  and  Nuttall,  148 

Irons,  654 

Irons  and  Graham,  44,  821 

Israel,  803,  806,  809 

IssaefF,  149 

Itzerott  and  Niemann,   364,   620, 

623,  625,  761 
Iwanow,  405 


JACKSON,  573,  66 1 

Jacob,  670 

Jacobsohn  and  Pick,  425 

Jacoby,  147 

Jadkewitsch,  368 

Jager,  293,  424 

Jamieson  and  Johnston,  772 

Jasuhara  and  Ogata,  117,  410 

Jenner,  no,  113,  197,  315,  558,  568 

Jez,  648 

Jobling  and  Flexner,  430 

Jochmann  and  Kraus,  488 

Jodasshon,  798 

Johnson,  665 

Johnston  and  Jamieson,  772 

Joos,  150 

Jordan,  293,  366,  367,  649,  664 

Jordan,  Russell,  and  Zeit,  636 

Jorgensen,  68 

Justinian,  581 

KAENSCHE,  69 
Kamen,  396 
Kanthack,  813 
Kaplan,  330 

Kaposi  and  Kundrat,  828 
Karlinski,  341,  368,  675 
Kartulis,  372,  373,  446,  688,  689, 
691 


Kashida,  654 

Kastle,  Rosenau,  and  Lumsden,  639 

Kazarinow,  702 

Keen,  641 

Kerr,  MacNeal,  and  Latzer,  84 

Kimla,  Poupe,  and  Vesley,  723 

Kinghorn  and  Todd,  550 

Kircher,  18,  22 

Kirchner,  439,  441 

Kitasato,  26,  116,  117,  208,  266, 
273,  385,  386,  388,  389,  394,  395, 
495,  58i,  583,  584,  585,  589,  596, 
6n,  612,  688,  701 

Kitasato  and  Behring,  160 

Kitasato  and  Weil,  265 

Kitasato  and  Yersin,  26 

Kitt,  114 

Klebs,  24,  451,  612,  687,  711,  736, 
742 

Klebs  and  Pasteur,  125 

Klein,  587,  591,  592 

Kleine,  562 

Klemperer,  502 

Klemperer  and  Bittu,  757 

Klemperer  and  Levy,  513,  636,  638 

Klemperer  (G.  and  F.),  505 

Klencki,  670 

Klimenko,  490,  707,  708 

Knapp,  475 

Knapp  and  Novy,  549,  550,  552, 
553 

Knisl,  622 

Knorr,  390,  595 

Kny,  47 

Koch,  23,  25,  26,  58,  125,  214,  217, 
235,  236,  242,  246,  247,  249,  266, 
269,  287,  303,  350,  368,  374,  400, 
401,  407,  409,  446,  447,  528,  549, 
551,  562,  601,  604,  605,  606,  607, 
611,  612,  613,  614,  622,  627,  631, 
632,  689,  691,  710,  711,  712,  713, 
715,  721,  722,  729,  731,  737,  738, 
739,  740,  742,  743,  744,  745,  746, 
748,  750,  751,  788,  795 

Koch  and  Gaffky,  688 

Kohlbrugge,  84 

Kolisko  and  Paltauf,  463 

Kolle,  181,  594,  614 

Kolle  and  Frosch,  357 

Kolle  and  Otto,  349 

Kolle  and  Pfeiffer,  638,  646,  648 

Kolle  and  Wassermann,  42,  45,  48, 
131,  430,  485,  520,  534,  536,  538, 
547,586,763,765,830,831 

Koplik,  488 

Korn,  760 

|  Kossee  and  Overbeck,  690 
I  Kossel,  117,  118,  163,  169,  368 
,  Krai,  831 


840 


Bibliographic  Index 


Krannhals,  368 

Kraus,  119,  140,  146,  147,  436 

Kraus  and  Buswell,  648 

Kraus  and  Jochmann,  488 

Krauss,  347 

Krefting,  442 

Kronig,  211 

Kronig  and  Menge,  383 

Kronig  and  Paul,  213 

Kruger,  62 

Krumwiede  and  Park,  752 

Kruse,  94,  369,  370,  399,  557,  602, 

667,  689,  699,  702 
Kruse  and  Pasquale,  688 
Kubel  and  Tiemann,  657 
Kuhne,  777 

Kundrat  and  Kaposi,  828 
Kurloff,  488 
Kurth,  352,  355 
Kutcher,  778 
Kutschbert-and  Neisser,  476 


LAENNEC,  732 

Lafleur  and  Councilman,  27,  688, 

689,  690,  691 
Laitinen,  434 
Lamar  and  Meltzer,  499 
Lamb  and  Birt,  522 
Lambert,  391,  503 
Lambl,  688,  689 
Lammershirt,  478 
Lamon  and  Meltzer,  511 
Landois,  163 
Landsteiner,  121 
Langenbeck,  484,  803 
Laplace,  213 
Larkin,  43 

Lartigau,  363,  368,  735 
Laschtschenko,  728 
Lassar,  787 
Latapie,  165,  272,  273 
Latour,  20 

Latour  and  Schwann,  20 
Latzer,  MacNeal,  and  Kerr,  84 
Laurent,  485 
Laveran,   27,   525,   526,   527,   531, 

532,  546,  558 
Laveran  and  Mesnil,  555,  556,  559, 

566,  567 

La  Wall  and  Leffmann,  231 
Lazear,  576,  577 
Lazear,  Carroll,  Reed,  and  Agra- 

monte,  576 
Leach,  90 
Leber,  50,  346 
Le  Dantec,  385 
Ledderhose,  366 
Leffmann  and  La  Wall,  231 


Leger  and  Duboscq,  707 

Lehman  and  Neumann,  278,  281 

Leichtenstern,  423 

Leishman,  197,  289,  307,  315,  316, 
558,  566,  567,  568,  569 

Leishman  and  Donovan,  27 

Lemoine,  355 

1'Engle  and  McFarland,  316 

Lenglet,  445 

Lennholm  and  Miller,  68 

Le  Noir,  368 

Lentz,  700 

Leo,  1 02,  784 

Lepierre,  428 

Lepp  and  Babes,  117,  422 

Lesage,  292,  368,  672 

Le  Sours  and  Griffon,  443 

Leubarth,  355 

Leuchs  and  Von  Lingelsheim,  429 

Leumann,  596 

Leumann  and  Hankin,  588 

Levaditi,  315,  550,  790 

Levaditi  and  Mamouelian,  792 

Levaditi  and  Mclntosh,  788,  793 

Levaditi  and  Nattan-Larrier,  802 

Levaditi  and  Yamanonchi,  320 

Levene,  737 

Levin,  357 

Levy,  501,  742 

Levy  and  Klemperer,  513,  636,  638 

Levy  and  Steinmetz,  779 

Lewis,  27 

Libman,  355,  665 

Liborius,  262,  264 

Liborius  and  Hansen,  261 

Liborius  and  Hesse,  265 

Lichtowitz,  477 

Liebig,  20 

Ligniere,  741 

Limbourg,  660 

Lincoln  and  McFarland,  506 

Lindemann,  121 

Linossier,  485 

Linton  and  Thomas,  558 

Lisbon,  593 

Lister,  25,  209 

Livingstone,  560 

Lock  wood,  210 

Lofner,  26,  182,  189,  190,  236,  237, 
379,  399,  409,  426»  432,  45i,  452, 
453,  454,  457,  460,  469,  474,  475, 
477,  480,  481,  514,  615,  628,  633, 
634,  660,  664,  672,  684,  685,  776, 
780 

Loffler  and  Abel,  638,  648 

Loiner  and  Shutz,  26,  601,  775,  781 

Longcope,  665 

Longcope  and  Fox,  493 

Lord,  440,  441 


Bibliographic  Index 


841 


Losch,  27,  688,  689,  690,  691,  695 
Losch  and  Hiickel,  48 
Losener,  645 
Low,  644 

Low  and  Emmerich,  77,  367 
Lubarsch,  129,  409,  675 
Lubbert,  214 
Lubenau,  357 
Lubinski,  399 
Lugol,  183 
Liihe,  531 

Lumsdcn,    Rosenau,    and    Kastle, 
639 


MACCOLLUM,  527,  531 

MacConkey,  66 1,  662 

Macfadyen,  638,  648 

Macfadyen  and  Rowland,  90,  638 

Macgregor,  687 

Mackie,  562 

MacNeal,  Latzer,  and  Kerr,  84 

Madsen,  26,  120,  121,  131,  160 

Madsen  and  Noguchi,  162 

Madsen  and  Salmonson,  139 

Maffucci  and  diVestea,  747 

Mafucci,  754 

Magendie,  122 

Maggiora,  81,  368,  688 

Maher,  670,  714 

Malassez  and  Vignal,  760 

Malfadyen,  498 

Mallory,  186,  642,  698 

Mallory  and  Wright,  197,  199,  424, 

425 
Mallory,  Councilman,  and  Pearce, 

467 

Malmsten,  704,  705,  824 
Malvoz,  150 

Mamouelian  and  Levaditi,  792 
Mann,  421 
Mannatti,  347 
Manson,  453,  525,  526,  527,  531, 

542,  554,  557,  570,  708 
Maragliano,  746,  747 
Marburg,  334 
Marchiafava,  525 
Marchiafava  and  Celli,  423 
Marchoux,  411 

Marchoux  and  Salimbeni,  546 
Marie,  422 

Marie  and  Morax,  392 
Marino,  197,  198,  199,  315,  558 
Marmier,  406 
Marmorek,  97,  356,  357,  359,  503, 

506 

Marshall  and  Gelston,  90 
Martha,  368 
Martin,  155,  162,  366,  406 


I  Martin  and  Meyer,  552 

Martin  and  Roux,  130 

Martin,  Fulleborn,  and  Mayer,  548 

Marx,  175 
j  Masselin  and  Thoinot,  598,  633 

Matschinsky  and  Rymowitsch, 
447,  449 

Matterstock,  757 

Matterstock  and  Doutrelepont,  757 

Mattson,  122 

Matzenauer,  478 

Maurea  and  Germano,  645 

Mayer,  Fulleborn,  and  Martin,  548 

McBryde  and  Bolton,  679 

McBryde,  Dorset,  and  Niles,  679 

McCarthy  and  Ravenel,  419,  420 

McClintock,  68 1 

McCollum  and  Burroughs,  472 

McConkey,  703 

McConnell,  772 

McCoy  and  Smith,  590 

McDaniel  and  Wilson,  469 

McFadyen,  753,  779 

McFarland,  310,  319,  411,  437,  467, 
472,  473,  479,  732,  747 

McFarland  and  1'Engle,  316 

McFarland  and  Lincoln,  506 

McFarland  and  Small,  73 

Mclntosh  and  Levaditi,  788,  793 

Mclntyre,  90 

McNeal,  569 

McNeal  and  Novy,  558,  565 

Megnin,  560 

Meier  and  Forges,  320 
i  Meirowsky,  798 

Melcher  and  Ortmann,  770 

Meltzer  and  Lamar,  499 
|  Meltzer  and  Lamon,  511 
I  Menge  and  Kronig,  383 

Mense,  567 

Mesnil,  127,  571 

Mesnil  and  Laveran,  555,  556,  559, 
566,  567 

Messea,  35 

Metalnikoff,  165 

Metschnikoff,  26,  105,  107,  120, 
121,  125,  126,  127,  128,  129,  130, 
141,  144,  145,  149,  150,  169,  307, 
732,  788 

Metschnikoff  and  Funck,  121 
i  Metschnikoff  and  Metalnikoff,  121 

Metschnikoff  and  Roux,  787 

Meunier,  63 
I  Meyer,  269,  544 

Meyer  and  Bergell,  649 

Meyer  and  Martin,  552 

Meyer  and  Ransom,  392,  394 

Meyers,  119,  180 

Michel,  454 


842 


Bibliographic  Index 


Migula,  32,  35,  38,  39,  40,  41,  84, 

278,  280,  281,  282,  493,  508,  666, 

685,  712,  786 
Mikylicz,  786 
Miller,  82,  83,  308,  309,  310,  311, 

312,  313,  314,  315,  478,  546,  631, 

640, 

Miller  and  Clarke,  211 
Miller  and  Lennholm,  68 
Milne  and  Ross,  547,  551 
Miquel,  286 
Mitchell,  24 

Mitchell  and  Stewart,  163 
Mithridates,  116 
Mittman,  81 
Moeller,  188,  757,  758 
Mohler  and  Eichhorn,  779,  783,  784 
Monnier,  368 

Montesano  and  Montesson,  396 
Montesson  and  Montesano,  396 
Montgomery,  87,  819,  820,  822 
Montgomery  and  Walker,  820 
Monti,  499 

Moore  and  Taylor,  779 
Morax,  446,  447,  448,  449,  450 
Morax  and  Elmassian,  467 
Morax  and  Marie,  392 
Morgenroth,  119,  131,  141,  147 
Morgenroth  and  Ehrlich,  120,  122, 

131,  163,  166,  322 
Mori,  508 
Moriya,  751 
Moro,  128 
Morro,  741 
Morse,  347 
Moschowitz,  397 
Moser,  359 
Moses,  762 
Mosso,  163 
Motz,  368 
Mouton,  127 
Much,  713,  714 
Muhlens,  793 
Muir  and  Ritchie,  184,   188,  193, 

555 

Muller,  179,  259 
Murphy,  809 
Murray,  551 
Musgrave  and  Clegg,  692,  694,  695, 

696,  812 

Musgrave  and  Strong,  699,  708 
Myers,  118,  120,  121,  131,  147 


NABARRO  and  Bruce,  557,  561 
Nabarro,  Bruce,  and  Greig,  561 
Nattan-Larrier  and  Levaditi,  802 
Neelow,  88 
Negri,  420,  421,  422 


Neisser,  26,  431,  441,  453,  475,  477, 

631,  798 

Neisser  and  Bruck,  798 
Neisser  and  Kutschbert,  476 
Neisser  and  Sachs,  171 
Neisser  and  Wechsberg,  167,  168, 

347,  349 
Neisser,  Wassermann,  and  Bruck, 

318,  319,  320,  322 
Nelis  and  Van  Gehuchten,  419 
Nepveu,  555 
Nessler,  75 
Netter,  501,  502 
Neufeld,  504 

Neuman  and  Swithinbank,  297 
Neumann,  368,  469,  488 
Neumann  and  Lehman,  278,  281 
Newman,  194 

Newman  and  Swithinbank,  726 
Newmark,  334 
Newsholme,  639 
Nicati  and  Rietsch,  612,  613 
Nicholas,  798 
Nicholas  and  Descos,  87 
Nicholls,  87 

Nichols  and  Schmitter,  263,  264 
Nicolaier,  26,  385 
Nicolas  and  Descos,  729 
Nicolaysen,  435 

Nicolle,  185,  442,  478,  571,  770 
Nicolle  and  Comte,  546,  571 
Niemann  and  Itzerott,   364,   620, 

623,  625,  761 

Niles,  Dorset,  and  McBryde,  679 
Nisot,  462 
Nissen,  129,  214 
Nissen  and  Behring,  129 
Nobe,  798 

Nocard,  43,  395,  397,  665,  677,  722 
Nocard  and  Railliet,  560 
Nocard  and  Roux,  234,  722 
Nocht  and  Behring,  213 
Noguchi,   27,    162,  320,  322,  326, 

327,  335,  336,  337,  338,  788,  793, 

794,  795,  798,  799 
Noguchi  and  Flexner,  162,  163,  390 
Noguchi  and  Madsen,  162 
Noisette,  487 
Nolen  and  Hewlett,  469 
Nolf,  147 
Norris,  43 

Norris  and  Oliver,  72 
Norris,  Pappenheimer,  and  Flour- 

noy,  550 
Novy,  177,  260,  261,  277,  547,  548, 

569,  680,  757 
Novy  and  Knapp,  549,  550,  552, 

553 
Novy  and  McNeal,  558,  565 


Bibliographic  Index 


843 


Novy  and  Vaughan,  68,  298 
Nuttall,    127,    128,    148,    174,   175, 

407,  592,  717 
Nuttall  and  Buchner,  128 
Nuttall  and  Inchley,  148 
Nuttall  and  Welch,  377,  379,  382 

OBERMEIER,  24,  26,  546 

Oertel,  464 

Oetinger,  368 

Ogata,  583,  589,  592,  687 

Ogata  and  Jasuhara,  117,  410 

Ogston,  343,  350 

Ohlmacher,  457,  474,  644,  718 

Oliver  and  Norris,  72 

Olsen,  485 

Ophuls,  44,  819 

Oppenheim,  334 

Oppler-Boas,  83 

Oriste-Armanni,  60 1 

Orlowski  and  Palmirski,  461 

Orth,  185 

Ortmann,  497 

Ortmann  and  Melcher,  770 

Oshida,  414 

Osier,  687,  688,  689 

Otto,  122 

Otto  and  Kolle,  349 

Overbeck  and  Kossee,  590 

Ovid,  17 

Oviedo,  800 

PALMIRSKE  and  Orlowski,  461 

Paltauf,  48 

Paltauf  and  Kolisko,  463 

Pane,  506 

Panfili,  213 

Pansini,  368 

Pappenheim,  717 

Pappenheimer,  Norris,  and  Flour- 

noy,  550 
Paquin,  746 
Pariette,  657 
Park,  97,  266,  388,  426,  427,  452, 

453,  470,  473,  474 
Park  and  Dunham,  699 
Park  and  Krumwiede,  752 
Park,  Briggs,  and  Beebe,  468 
Parke  and  Williams,  493 
Parker,     Rosenau,     Francis,     and 

Beyer,  580 

Pasquale  and  Kruse,  688 
Passet,  343,  350,  666 
Passler,  506 
Pasteur,   19,  20,  25,  26,   102,   113, 

114,  135,  260,  264,  374,  407,  409, 

410,  412,  413,  416,  417,  419,  420, 

492,  598,  600 


Pasteur  and  Chevreul,  21 

Pasteur  and  Klebs,  125 

Pasteur  and  Toussaint,  598 

Pasteur- Chamberland,  207,  208 

Paterson,  747 

Patton,  570 

Paul  and  Kronig,  213 

Paulicki,  754 

Pawlowski,  63,  724 

Pawlowsky,  616,  618 

Peabody  and  Pratt,  641,  650,  660 

Pearce,  355,  463,  464 

Pearce,  Councilman,  and  Mallory, 

467 

Pearson,  781 

Pearson  and  Gilliland,  753 
Pease  and  Bolton,  62 
Peckham,  671 
Perkins,  368,  510 
Perkins  and  Howard,  359,  360 
Pernoss  and  Ferni,  391 
Perroncita,  664 
Perroncito,  598 
Peterson,  442 
Petkowitsch,  656 
Petri,  246,  249,  285,  475,  760 
Petruschky,  41,  43,  240,  356,  643, 

645,  664,  676,  740 
Pfeiffer,    120,    140,    141,    142,    166, 

181,  234,  271,  514,  515,  516,  517, 

518,  519,  616,  625,  627,  628,  629, 

760,  761 

Pfeiffer  and  Beck,  517 
Pfeiffer  and  Canon,  26 
Pfeiffer  and  Frankel,  346,  351,  375, 

386,  387,  388,  400,  402,  403,  459, 

503,  599,  607,  621,  626,  711,  725, 

776 

Pfeiffer  and  Kolle,  638,  646,  648 
Pfeiffer  and  Vogedes,  616 
Pfuhl,  372,  738 
Phisalix  and  Bertrand,    117,    118, 

161 

Pianese,  571 
Piatkowski,  720 
Pick  and  Jacobsohn,  425 
Picq  and  Chenot,  784 
Pictet  and  Yung,  65 
Pierce,  502 
Piorkowski,  655,  673 
Pitfield,  191,  392,  68 1 
Platania,  102 

Plaut,  48,  478,  479,  485,  487,  831 
Plencig,  23 
Plett,  no 
Pohl,  293 
Pollender,  24 

Pollender  and  Davaine,  25 
Pope,  648 


844 


Bibliographic  Index 


Forges  and  Meier,  320 

Portier  and  Richet,  122 

Posadas  and  Wernicke,  819 

Pott,  63 

Poupe,  Kimla,  and  Vesley,  723 

Pratt  and  Peabody,  641,  650,  660 

Preindelsberger,  81 

Prescott,  68,  666 

Prescott  and  Winslow,  239 

Prior  and  Kinkier,  619,  620,  621, 

622,  623,  631 
Proca  and  Babes,  747 
Proskauer  and  Beck,  59,  726 
Prowazek,  372,  559 
Prowazek  and  Hoffmann,  799 
Prudden,  289,  354,  731 
Prudden  and  Hodenpyl,  749 


QUENU  and  Chauffard,  398 
Quincke,  830 
Quincke  and  Roos,  688 
Quinquaud,    485 


RABINOWITSCH,  297,  760,  818 

Railliet  and  Nocard,  560 

Ramon,  392 

Ransom  and  Meyer,  392,  394 

Rappaport,  589 

Raskin,  355 

Ravenel,  64,  87,  233,  237,  238,  243, 
261,  292,  293,  295,  729,  750 

Ravenel  and  McCarthy,  419,  420 

Redi,  1 8 

Reed,  576 

Reed  and  Carroll,  576,  580 

Reed,  Carroll,  and  Agramonte,  577 

Reed,  Carroll,  Lazear,  and  Agra- 
monte, 576 

Reed,  Vaughan,  and  Shakespeare, 

639 

Reichel,  208,  723 
Reichel  and  Kngle,  421 
Remak,  828 
Remy,  653 
Ress,  485 
Rettger,  85,  410 
Reyes,  470 
Ribbert,  347,  349 
Richardson,  643,  644,  652 
Richet  and  Portier,  122 
Ricketts,  819 
Rideal,  214 

Rideal  and  Walker,  305 
Riedel,  606 
Rieder,  63 
Rieger,  424 
Rietsch  and  Nicati,  612,  613 


Rindfleisch,  627 

Rist,  461 

Ritchie  and  Muir,   184,   188,   193, 

555 

Ritter,  488 
Rivolta,  27,  754 
Robb,  340,  341 
Robb  and  Ghirskey,  81 
Robb  and  Welch,  210 
Robertson,  637 
Robey  and  Ernst,  150 
Robin,  484,  485 
Robinson,  218 
Rodet,  349 

Roger,  98,  loo,  410,  487 
Roger  and  Charrin,  102,  149,  760 
Roger,  Cadio,  and  Gilbert,  754 
Rogers,  438,  566,  569,  570 
Rogone,  211 
Rolleston,  670 
Roloff,  754 
Romanowsky,   197,   199,  550,  558, 

788 

Roos  and  Quincke,  688 
Rosenau,   124,   159,  218,  472,  589, 

686,  724,  727 

Rosenau  and  Anderson,  123,  161 
Rosenau,    Lumsden,    and    Kastle, 

639 
Rosenau,     Parker,     Francis,     and 

Beyer,  580 

Rosenbach,  343,  349,  350,  361 
Rosenberger,  250 
Rosenow,  502,  506 
Roser,  125 

Ross,  200,  526,  527,  566,  567,  577 
Ross  and  Milne,  547,  551 
Rossi,  193 
Rost,  766,  774 
Rothberger,  654 
Roudoni  and  Sachs,  337 
Rouget  and  Vaillard,  396 
Roux,  26,  116,  156,  204,  260,  264, 

269,  409,  453,  475,  477,  485 
Roux  and  Borrel,  391,  398 
Roux  and  Chamberland,  116,  377, 

412 

Roux  and  Martin,  130 
Roux  and  Metschnikoff,  787 
Roux  and  Nocard,  234,  722 
Roux  and  Yersin,  91,  1 16,  460,  461, 

464 

Rowland  and  Macfadyen,  90,  638 
Rudolph,  767 
Ruediger,  354,  505 
Ruffer,  616 
Rumpf,  648 
Ruppel,  736,  737 
Russell,  702 


Bibliographic  Index 


845 


Russell  and  Evans,  218 
Russel  and  Hiss,  699 
Russell,  Jordan,  and  Zeit,  636 
Russo-Travali  and  Blasi,  464 
Ruzicka,  365 

Rymowitsch     and     Matschinsky, 
447,  449 


SABOURAUD,  824,  825,  826,  829,  831 

Sabrazes,  478 

Sacharoff,  546 

Sachs  and  Neisser,  171 

Sachs  and  Roudoni,  337 

Salant,  103 

Salimbeni  and  Marchoux,  546 

Salkowski,  73,  669 

Salmon,  602,  603,  664,  678,  750 

Salmon  and  Smith,  116,  601,  678, 

679 

Salmonson  and  Madsen,  139 
Salomonsen,  266 
Salsano  and  Fermi,  756 
Sambon,  561 
Sanarelli,  64,  576,  577,  664,  682, 

683,  684 
Sander,  724 
Sanfelice,  369,  399,  818 
Sattler,  477 
Savage,  654 
Scala,  699 
Schaudinn,  547,  565,  689,  690,  691, 

693,  695 
Schaudinn  and  Hoffmann,  26,  82, 

787,  788,  799,  80 1 
Scherer,  424,  426 
Schereschewsky,  793 
Schering,  179,  215 
Schick  and  von  Pirquet,  122 
Schleich,  477 
Schmidt,  424 

Schmitter  and  Nichols,  263,  264 
Schneider,  214,  424 
Schonlein,  828 
Schottelius,  609 
Schottmuller,  353,  354,  359 
Schroder,  501 

Schroder  and  Van  Dusch,  25,  204 
Schroter,  293 
Schuffner,  537 
Schulze,  19 

Schumburg  and  Gauss,  35 
Schutz   and    Loffler,  26,  601,  775, 

781 

Schutze  and  Wassermann,  119,  148 
Sch waive,  401 
Schivam  and  Latour,  20 
Sedgwick,  285,  286,  661 
Sedgwick  and  Tucker,  286 


Sedgwick  and  Winslow,  65,  637 
Seifert,  439 
Selter,  241,  784 
Semmelweiss,  23 
Semple  and  Wright,  646 
Shakespeare,  610,  621,  624 
Shakespeare,  Vaughan,  and  Reed, 

639 

Shaw,  97 

Shiga,  26,  688,  689,  699,  702,  704 
Sholly  and  Goodwin,  430 
Sievenmann,  50 
Siffer,  McClintock,  and  Boxmeyer, 

68 1 

Silber,  299 
Silverschmidt,  372 
Simon,  357 
Simpson,  582,  587 
Sjoo  and  Tornell,  720 
Small  and  McFarland,  73 
Smirnow,  62 
Smith,  192,  228,  230,  291,  461,  5.1 1, 

643,  664,  673,  679,  680,  681,  722 
Smith  (L.)»  194.  237 
Smith  (T.),  70,  122,  151,  156,  241, 

266,  390,  662,  723,  748,  749,  750, 

754 

Smith  and  McCoy,  590 
Smith  and  Salmon,  116,  601,  678, 

679 

Smith  and  Weidman,  373 
Sobernheim,  612,  616 
Solowiew,  708 
Somers,  658 

Southard  and  Gay,  123,  124 
Sowade,  793 
Soyka,  58 
Spallanzani,  19 
Spengler,  488 
Spiller,  420 

Spronck,  460,  699,  766 
Starkey,  657,  658 
Steel,  430 
Steele,  84,  648 

Steinhardt  and  Bauzhaf,  160 
Steinhardt  and  Besredka,  124 
Steinmetz  and  Levy,  779 
Stern,  130,  648,  789,  797 
Sternberg,  125,  301,  303,  346,  353, 

368,  389,  492,  576,  611,  636,  633 
Sternberg  and  Bitter,  70 
Stewart,  178,  384 
Stewart  and  Baldwin,  383 
Stewart  and  Mitchell,  163 
Sticker,  50,  771 
Stiles,  690 

Stimson,  414,  415,  416 
Stitt,  542 
Stokes,  296,  654 


846 


Bibliographic  Index 


Stokes  and  Gilchrist,  818,  821 

Stokvis  and  Winogradow,  708 

Stooss,  486 

Strasburger,  84 

Straus,  778 

Strehl,  222 

Strong  and  Musgrave,  699,  708 

Stiihlerm,  508 

Sugai,  770 

Surveyor  and  Boyce,  813,  817 

Swithinbank  and  Neuman,  297 

Swithinbank  and  Newman,  726 

Szemetzchenko,  488 


TAKAKI  and  Wassermann,  118,  391 

Tangl,  461 

Tashiro,  770 

Taube  and  Weber,  756 

Tavel,  47,  359,  399,  636 

Tavel  and  Alvarez,  757 

Taylor  and  Moore,  779 

Tchistowitch,  119,  146,  499 

Tedeschi,  783,  798 

Telamon,  492 

Terre  and  Bugarre,  756 

Theiler,  546 

Thelling,  744,  746 

Theodoric,  22 

Thiercelin,  368 

Thoinot  and  Masselin,  598,  633 

Thomas,  114 

Thomas  and  Linton,  558 

Thompson,  696 

Thompson- Yates,  556,  661  r 

Tinctin,  550 

Tidswell,  593 

Tiemann  and  Kubel,  657 

Timpe,  228 

Tizzoni  and  Centanni,  746 

Todd,  555 

Todd  and  Button,  547,  551,  557 

Todd  and  Kinghorn,  550 

Todd,  Button,  and  Christy,  557 

Tornell  and  Sjoo,  720 

Torrey,  437,  438 

Torrey  and  Buxton,  126 

Toussaint,  409 

Toussaint  and  Pasteur,  598 

Trambusti,  65 

Traube  and  Gscheidel,  127,  166 

Trendenburg,  355 

Treskinskaja,  61 

Triboulet,  368 

Trillat,  218 

Trudeau,  744 

Trudeau  and  Baldwin,  742,  744 

Tsiklinsky,  64 

Tsugitani,  694 


Tucker  and  Sedgwick,  286 
Tunnicliff,  479,  480,  481,  482 
Tyndall,  1 8,  19,  58 


,  462 
Uhlenhuth,  147 
Uhlenhuth  and  Xylander,  720 
Ukke  and  Grigorjeff,  377 
Unna,  81,  442,  443,  719,  791,  792 
Uschinsky,  461 


VAIU,ARD  and  Bopter,  702 

Vaillard  and  Rouget,  396 

Valagussa,  688 

Van  de  Velde,  347,  359 

Van  de  Velde  and  Benys,  349 

Van  Busch  and  Schroeder,  25,  204 

Van  Ermengem,  192,  299,  612,  613, 

790 

Van  Gehuchten  and  Nelis,  419 
Van  Helmont,  18 
Van  Leeuwenhoek,  18,  20,  29 
Van  Steenberghe  and  Grysez,  87 
Varro,  21 

Vaughan,  90,  124,  298,  558,  638 
Vaughan  and  Buxton,  151 
Vaughan  and  Cooley,  669 
Vaughan  and  Novy,  68,  398 
Vaughan,  Reed,  and  Shakespeare, 

639 

Veasy  and  de  Schweinitz,  448,  449 
Vedder  and  Buval,  700 
Veillon,  479 
Verneuil,  385 

Vesley,  Kimla,  and  Poupe,  723 
Viereck,  690,  693 
Vierordt,  608 

Vignal  and  Arustamoff,  42 
Vignal  and  Malassez,  760 
Villemin,  711 
Villiers,  612 
Vincent,  478,  479,  483,  812,  813, 

814 

Vincentini,  82 
Vincenzi,  488 
Viola  and  Bonome,  62 
Viquerat,  349,  746 
Virchow,  740,  771 
Virgil,  17 

Vogedes  and  Pfeiffer,  616 
Voll,  150 

Volpino  and  Bertarelli,  790 
Von  Bungern,  119,  120,  121,  140, 

165 

Von  Frisch,  785 
Von  Ley  den,  649 
Von  Lingelsheim,  346,  351,  424 


Bibliographic  Index 


847 


Von  Lingelsheim  and  Leuchs,  429 

Von  Mayer,  399 

Von  Pirquet,  740,  741,  797 

Von  Pirquet  and  Schick,  122 

Von  Szekely,  37 

Vuillemin,  485 

WADSWORTH,  499,  504 

Wagner,  102 

Walger,  648 

Walker,  145 

Walker  and  Montgomery,  820 

Walker  and  Dideal,  305 

Walz  and  Baumgarten,  745 

Warren,  230,  457 

Wasdin  and  Geddings,  577 

Washbourn,  503,  506 

Wassermann,  26,  n 8,  147,  171,  318, 
319,  322,  324,  326,  328,  330,  331, 
333,  334,  335,  336,  338,  367,  434, 
435,  437,  797,  799 

Wassermann  and  Bruck,  797 

Wassermann  and  Citron,  328 

Wassermann  and  Kolle,  42,  45,  48, 
131,  430,  485,  520,  534,  536,  538, 
547,  586,  763,  765,  830,  831 

Wassermann  and  Schiitze,  119,  148 

Wassermann  and  Takaki,  118,  391 

Wassermann,  Neisser,  and  Bruck, 
318,  319,  320,  322 

Weaver,  479 

Weber  and  Traube,  756 

Wechsberg  and  Neisser,  167,  168, 

347,  349 

Weeks,  446,  447,  448,  477 
Weibel,  631 
Weichselbaum,  293,  423,  424,  425, 

426,  429,  430,  492,  501,  502,  644 
Weichselbaum    and    Frankel,    88, 

373,  492,  509 
Weidman  and  Smith,  373 
Weigert,  25,  137,  172,  174,  403,  454, 

494,  495,  719,  764,  806,  815,  817 
Weigert  and  Gram,  185 
Weil  and  Kitasato,  265 
Weinzirl,  61 
Welch,  81,  131,  145,  341,  377,  384, 

397,  454,  472,  494 
Welch  and  Flexner,  377,  383,  464 
Welch  and  Nuttall,  377,  379,  382 
Welch  and  Robb,  210 
Wellenhof,  474 
Welsh,  340 

Wernich  and  Chauveau,  125 
Wernicke,  26,  589,  631 
Wernicke  and  Posadas,  819 
Wertheim,  431,  433,  434 
Wesbrook,  452,  454,  469,  657 
Wesbrook  and  Hankin,  406 


Wesenberg,  372 

Wheeler,  90 

Widal,  149,  633,  640,  644,  649,  650, 

653,  673 
Widal  and  Chantemesse,  647,  648, 

699 

Widal  and  Griinbaum,  649 
Wiener  and  Gruber,  616 
Wiens,  670 
Wigura,  81 

Willcomb  and  Winslow,  288 
Williams,  368,  383,  462,  695,  792 
Williams  and  Lowden,  421 
Williams  and  Parke,  493 
Wilson,  588,  589 
Wilson  and  McDaniel,  469 
Windsor  and  Wright,  522 
Winkler,  259 

Winogradow  and  Stokvis,  708 
Winogradsky,  74 
Winslow,  8 1 

Winslow  and  Prescott,  239 
Winslow  and  Sedgwick,  65,  637 
Winslow  and  Willcomb,  288 
Winterbottom,  554 
Winterintz  and  Doderlein,  85 
Witte,  228,  229,  241,  505,  653,657, 

659,  662 

Wladimiroff,  549 
Wolf,  502 
Wolff,  650,  806 
Wolff-Eisner,  742,  798 
Wolffhugel,  287,  288,  606 
Wolfhugel,  287,  288 
Wollstein,  490,  519,  700 
Wollstein  and  Frankel,  491 
|  Wood,  177,  507 
Wood  and  Hiippe,  411 
Woodhead,  36,  809 
Woodward,  687 
Wright,  1 15,  197,  264, 266,  267,  287, 

289,  292,  293,  308,  311,  312,  315, 

317,  349,  355,  434,  438,  522,  558, 

568,  572,  638,  646,  647,  745,  783, 

807,  815,  816,  817 
Wright  (J.  H.),  573 
Wright  and  Brown,  804,  805 
Wright  and  Douglas,  127,  307,  316 
Wright  and  Mallory,  197,  199,  424, 

425 

Wright  and  Semple,  646 
Wright  and  Windsor,  522 
Wschinsky,  669 
Wunschheim  and  Fischel,  130 
Wiirtz,  653,  668,  675 
Wyman,  583,  589 
Wynekoop,  519 
Wyssokowitsch     and     Zabolotny, 

591,  597 


848 


Bibliographic  Index 


XYLANDER  and  Uhlenhuth,  720 


YAMANONCHI  and  Levaditi,  320 
Yersin,  581,  582,  583,  584,  585,  588, 

589,  590,  59i,  592,  594,  595,  596, 

597 

Yersin  and  Kitasato,  26 
Yersin  and  Roux,  91,  116,  460,  461, 

464 

Young,  433,  435,  436 
Yung  and  Pictet,  65 


ZABOLOTNY    and    Wyssokowitsch, 

591,  597 
Zaufal,  501 


Zeit,  62,  782 

Zeit,  Jordan,  and  Russell,  636 

Zenker,  179,  186,  199 

Ziegler,  687 

Ziehl,  181,  189,  634,  716,  717 

Zieler,  186 

Zimmermann,  292,  293 

Zinno,  460 

Zinsser,  264,  511 

Zinsser  and  Hiss,  41,  225,  352,  362, 

424,  496,  497,  506,  632,  663,  777, 

822 

Zopf,  37,  293,  507 
Zuber,  479 
Zupinski,  260 
Zupnik,  393 
Zur  Nedden,  450 


INDEX 


ABBOTT'S  method  of  staining  spores, 

1 88 

Abscess  of  liver  in  amebic  dysen- 
tery, 697 
tuberculous,  731 
Acetic  fermentation,  67 
Achorion,  46 
schonleinii,  828 
cultivation,  830 

Krai's  method,  831 
pathogenesis,  831 
Acid,  carbolic,  as  disinfectant,  215 

tuberculinic,  737 
Acids  as  germicides,  213 

production  of,  71 
Actinodiastase,  127 
Actinomyces,  43,  803,  804 
bovis,  803 

cultivation,  806 
distribution,  804 
general  characteristics,  803 
lesions,  811 
morphology,  804 
pathogenesis,  809 
virulence,  809 
grain,  805 
madurae,  812 
cultivation,  814 
cultural  characteristics,  817 
general  characteristics,  812 
lesions,  814 
morphology,  813 
Actinomycosis,  803 

communication  of,  to  man,  809 
Acute     contagious    conjunctivitis, 

446 

Adami  and   Chapin's  method   for 
isolation  of  typhoid  bacillus,  656 
Addiment,  120,  144 
Adhesion  preparations,  256 
Aerobes,  59 
Aerogens,  66 
African  lethargy,  554 
Agar-agar  as  culture-media,  232 
bile-salt,  662 

blood,  as  culture-media,  234 
culture,  255 

glycerin,  as  culture-media,  234 
54 


Agar-agar,  litmus-lactose,  654 
preparation  of,  233 

Ravenel's  method,  233 
Agglutination,  149 

test,  Koch's,  for  tubercle  bacillus, 

746 

technic  of,  151 
Widal  reaction  of,  649 
Agglutinins,  140 
Agglutometers,  650 
Aggressins,  146 
Ague-cake,  540 
Air,  bacteria  in,  58,  283 

quantitative     estimation,     by 

Hesse's  method,  284 
by  Petri's  method,  285 
by  Sedgwick's  method, 

286 

bacteriology  of,  283 
displacement  of,  by  inert  gases, 

in  anaerobic  cultures,  260 
of  sick-room,  disinfection,  211 
withdrawal  of,  in  anaerobic  cul- 
tures, 260 
Air-examination,  Petri's  sand  filter 

for,  285 
Sedgwick's   expanded   tube   for, 

285 

Alcoholic  fermentation,  67 
Aleppo  boil,  572 
Alexin,  128,  144,  166 
Algid  cases  of  malaria,  539 
Alkali-albuminate,     Deycke's,     as 

culture- media,  237 
Alkalies,  production  of,  71 
Alkaline   blood-serum   as   culture- 
media,  237 
Allergia,  123 
Altmann's  syringes,  269 
Amboceptor  dose  in  Wassermann 

reaction,  326 

hemolytic,   for  Wassermann  re- 
action, 323 
unit    in    Wassermann    reaction, 

325,  326 

Amboceptors,   144,  145 
Amebadiastase,   127 
i  Amebae  and  suppuration,  372 

849 


850 


Index 


Amebic  dysentery,  689 

abscess  of  liver  in,  697 
lesions,  697 
American  trypanosomiasis,  564 

transmission,  565 
Amoeba  coli,  689 

rhizopodia,  690 
dysenteriae,  689 
kartulisi  as  cause  of  suppuration, 

372 

martinatalium  as  cause  of  suppu- 
ration, 373 
Anaerobes,  59 
facultative,  59 
optional,  59 
Anaerobic  bacteria,  cultivation  of, 

260 
by  absorption  of  atmospheric 

oxygen,  263 

by  displacement  of  air,  259 
by  exclusion  of  atmospheric 

oxygen,  265 

by  formation  of  vacuum,  260 
by  reduction  of  oxygen,  264 
by  withdrawal  of  air,  260 
cultures,  Botkin's  apparatus  for 

making,  262 
Buchner's  method  of  making, 

263 
Frankel's  method  of  making, 

261 

Hesse's  method  of  making,  265 
Koch's  method  of  making,  266 
Liborius'  tube  for,  261 
Nichols  and  Schmitter's  meth- 
od of  making,  263 
Novy's  jars  for,  261 
Salomonsen's  method  of  mak- 
ing, 266 
Wright's   method   of  making, 

264,  267 
Zinsser's   method   of   making, 

264 

Anaphylactin,  124 
Anaphylaxis,  122 

passive,  124 

Anesthetic  leprosy,  771,  773 
Angina,  Vincent's,  478 
Animal  holder,  Latapie's,  272,  273 

inoculations,  271 
Animalculae,  29 
Animals,     experimentation    upon, 

269 
method  of  securing  blood  from, 

273 

postmortems  on,  275 
Anjeszky's     method     of     staining 

spores,  1 88 
Anopheles  maculipennis,  541,  542 


Anthracin,  406 
Anthrax,  400 

bacillus  of,  400 

bacteriologic  diagnosis,  411 

causes  of  death  from,  409 

distribution,  400 

in  cattle,  how  acquired,  407 

lesions,  407 

means     of     protecting     animals 
against,  410 

Pasteur's   protective  inoculation 
against,  409 

sanitation  in,  411 

serum  therapy  in,  410 

vaccination  in,  409 
Antibiosis,  influences  on  growth  of 

bacteria,  63 

Antibodies,  miscellaneous,   163 
Anticholera  serum,  617 
Antiferments,  140 
Antiformin  for  isolation  of  tubercle 

bacillus,  720 
Antigen,  115 

syphilitic,  319 

titration  of,  328 
Antigonococcus  serum,  437 
Anti-immune  bodies,  169 
Antikorper,  140,  471 
Antimeningococcus  serum,  430 
Antiphthisin,  742,  743 
Antipneumococcus  serum,  505,  506 
Antirabic  serum,  422 
Antisepsis,  25 

early,  25 

Antiseptic  action,  results  of,  303 
Antiseptics,  201 

determination  of  value,  302 

influence  on  growth  of  bacteria, 

6.5  .  . 
inhibition  strengths  of,  216 

Antispermotoxin,   121 

Antistaphylococcus  serum,  349 

Antistreptococcus  serum,  359 

Antistreptokolysin,  357 

Antitoxin  of  diphtheria,  155,  471 
bleeding,  156 
effect  on  death-rate,  473 
immunization  of  animals,  156 
method  of  administration,  471 
paralysis  after  use  of,  472 
potency  of  serum,  157 
preparation  of  serum,  157 

of  toxin,  156 
prophylaxis,  471 
tetanus  after  use  of,  473 
treatment  with,  471 
tetanus,  160,  397 

Antitoxins,  153 

Antitubercle  serums,  746 


Index 


851 


Antityphoid  serums,  648 

Antivenene,  161 

Antivenomous  serum,  161 

Apilacao,  564 

Apparatus,   complete  leveling,   for 

pouring  plate  cultures,  247 
Hesse's,    for   collecting    bacteria 

from  air,  284 

Koch's,     for     coagulating     and 
sterilizing    blood-serum,    235, 
236 
Wolfhiigel's,  for  counting  colonies 

of  bacteria  upon  plates,  287 
Aqueous  solution  for  staining,  177 
Argas  miniatus,  546 
Arnold's  steam  sterilizer,  205 
Aromatics,  production  of,  73 
Arthrospores,  38 
Ascococcus,  39 
Asiatic  cholera,  604 

immunity  against,  616 

prophylaxis,  617 

rice-water  discharges  of,  613 

sanitation  in,  618 

specific  organism  of,  discovery, 

605 

Aspergillus,  48 
flavus,  50 
fumigatus,  49 
glaucus,  49 
malignum,  49 
nidulans,  49 
niger,  50 
subfuscus,  50 
Association,    influence    on    growth 

of  bacteria,  63 

Atmospheric    oxygen,     absorption 
of,  in  anaerobic  cultures,  263 
exclusion  of,  in  anaerobic  cul- 
tures, 265 

Auditory    meatus,    external,    bac- 
teria in,  82 
Autoclave,  206 

sterilization  in,  206 
Axenfeld,  bacillus  of,  448 


BABES  and  Cornil's  method  of  stain- 
ing spirillum  cholerae  Asiaticae, 
608 

Babes-Ernst  granules,  34 
Babes'  tubercles,  419 
Bacillary  dysentery,  699 
diagnosis,  704 
lesions,  702 
serum  therapy,  704 
emulsion,  745 
Bacilli,  paracolon,  665 
paratyphoid,  665 


Bacilli    resembling   Bacillus   diph- 

theriae,  474 
of  anthrax,  411 
typhosus,  663 

meat-poisoning  group,  665 
pneumonic  group,  665 
psittacosis  group,  665 
table    for    differentiation, 

664 

typhoidal  group,  665 
tetanus  bacillus,  399 
tubercle  bacillus,  756 
Bacillus,  39 

acidi  lactici,  666 
aerogenes  capsulatus,  377 
cultivation,  380 
distribution,  378 
morphology,  378 
pathogenesis,  382 
sources  of  infection,  383 
staining,  379 

Welch  and  Nuttall's  meth- 
od, 379 

vital  resistance,  382 
anthracis,  400 

avenues  of  infection,  406 

bacilli  resembling,  411 

cultivation,  403 

isolation,  403 

means  of  diminishing  virulence, 

409 

metabolic  products,  405 
morphology,  402 
pathogenesis,  406 
similis,  411 
staining,  403 
thermic  sensitivity,  405 
anthracoides,  411 
avicidum,  598 
avisepticus,  598 
Bordet-Gengou,  488 

and  influenza  bacillus,  differ- 
ences between,  491 
cultivation,  490 
isolation,  489 
metabolic  products,  490 
morphology,  489 
pathogenesis,  490 
staining,  489 
butter,  760 
butyricus,  760 
canal-water,  capsulated,  508 
capsulated  canal-water,  508 
capsulatus  mucosus,  507 
capsule,  509 
cavicida,  666 
choleras,  598 
gallinarum,  598 
cultivation,  598 


852 


Index 


Bacillus  choleras   gallinarum,  gen- 
eral characteristics,   598 
immunity  against,  600 
lesions,  600 

metabolic  products,  599 
morphology,  598 
pathogenesis,  600 
staining,  598 
vital  resistance,  599 
coli  communior,  669 
communis,  666 

bacillus  typhosus  and,  differ- 
entiation, 651 
cultural,  652 
serum,  651 
cultivation,  667 
diagnosis,  differential,  672 
distribution,  666 
general  characteristics,  666 
immunization  against,  672 
in  summer  infantile  diarrhea, 

671 
in  water,  291,  674 

MacConkey's  medium  for 

detecting,  66 1 
Wiirtz's  medium  for  de- 
tecting, 675 
influence  of  environment  on, 

671 

metabolic  products,  668 
morphology,  667 
pathogenesis,  669 
staining,  667 
toxic  products,  669 
virulence,  670 
vital  resistance,  668 
comma,  606 
cuniculicida,  598,  601 
diphtherias,  451 

bacilli  resembling,  474 
bacteria  associated  with,  464 
bacteriologic  diagnosis,  456 
chief  types,  469 
classification  of  types,  469 
contagion  from,  468 
cultivation,  454 

lyoffler's  method,  454 
differentiation  of,  from  pseu- 
dodiphtheria   bacillus,    468, 

474 

general  characteristics,   451 
metabolic  products,  460 
morphology,  451 
pathogenesis,  462 
relation  of,  to  diphtheria,  467 
seats  of  infection  by,  464,  465 
specificity,  467 
staining,  452 

Neisser's  method,  453 


Bacillus  diphtherias,  staining  with 
Lofner's  alkaline  methylene- 
blue,  452 

vital  resistance,  460 

Wesbrook's  types  of,  452,  469 
dysenteriae,  699 

cultivation,  700 

Flexner  variety,  702 

Hiss-Russel  variety,  702 

metabolic  products,  701 

morphology,  700 

pathogenesis,  702 

Shiga-Kruse  variety,  702 

staining,  700 

varieties,  702 

vital  resistance,  701 
enteritidis,  675 

cultivation,  675 

general  characteristics,  675 

lesions,  676 

morphology,  675 

pathogenesis,  676 

staining,  675 
faecalis  alkaligenes,  676 
fluorescens  liquefaciens,  365 
fusiformis,  478 

and  spirochaeta  Vincenti,   re- 
lation, 479 

cultivation,  480 

morphology,  480 

pathogenesis,  483 
Gartner's,  675 
geniculatus     in     carcinoma     of 

stomach,  83 
icteroides,  682 

cultivation,  682 

distribution,  682 

metabolism,  683 

morphology,  682 

pathogenesis,  683 

staining,  682 

vital  resistance,  683 
influenzas,  514 

and    Bordet-Gengou    bacillus, 
differences  between,  491 

cultivation,  516 

immunity  against,  517 

isolation,  515 

morphology,  514 

pathogenesis,  517 

pseudo-,  519 

specificity,  517 

staining,  514 

vital  resistance,  517 
Klebs-Loffler,  45 1 .    See  also  Ba- 
cillus diphtheria. 
lactis  aerogenes,  666 
leprae,  762 

cultivation,  765 


Index 


853 


Bacillus  leprae,  cultivation,  Clegg's 

method,  767 

Duval's  method,  767,  768 
Rost's  method,  766 
distribution,  762 
general  characteristics,  762 
lesions,  771 
morphology,  763 
pathogenesis,  770 
staining,  763 
mallei,  775 

cultivation,  779 
distribution,  776 
general  characteristics,  775 
immunity  against,  784 
isolation,  777 
metabolic  products,  780 

mallein,  781 
morphology,  776 
pathogenesis,  781 
staining,  776 

Kiihne's  method,  777 
Loffler's  method,  776 
virulence,  784 
vital  resistance,  777 
melitensis,  520.     See  also  Micro- 
coccus  melitensis. 
Moeller's  grass,  758 
neapolitanus,  666 
Nocard's,  677 
cedematis  maligni,  374 
cultivation,  374 
distribution,  374 
immunity  against,  377 
lesions,  376 

metabolic  products,  375 
morphology,  374 
pathogenesis,  376 
staining,  374 

of  anthrax,  400.     See  also  Bacil- 
lus anthracis. 
of  Axenfeld,  448 
of  Buffelseuche,  601 
of  chicken-cholera,  598.    See  also 

Bacillus  cholera  gallinarum. 
of  Ducrey,  442 
cultivation,  443 

Davis'  method,  443 
morphology,  443 
pathogenesis,  445 
staining,  443 
vital  resistance,  445 
of  Fasching,  507 
of  gaseous  edema,  377.     See  also 

Bacillus  aerogenes  capsulatus. 
of  hemorrhagic  septicemia,  597 
of  Hoffmann,  474.  See  also 

Bacillus,  pseudodiphtheria. 
of  Koch- Weeks,  446 


Bacillus  of   Koch- Weeks,  associa- 
tion, 448 
cultivation,  448 
morphology,  447 
pathogenesis,  448 
staining,  447 

of  malignant  edema,   374.     See 
also  Bacillus  cedematis  maligni. 
of  Morax-Axenfeld,  447 
cultivation,  449 
morphology,  449 
pathogenesis,  450 
staining,  449 

of  rabbit  septicemia,  598,  601 
of  Shiga,  699.     See  also  Bacillus 

dysenteric. 
of  swine-plague,  60 1 
of  syphilis,  787 

of  tetanus,  385.     See  also  Bacil- 
lus tetani. 
of  Weeks,  446.     See  also  Bacillus 

of  Koch-Weeks. 
of  Wildseuche,  60 1 
Oppler-Boas,    in    carcinoma    of 

stomach,  83 
pestis,  581 

cultivation,  585 

diagnosis,  593 

experimental    infection    with, 

590 

immunity  against,  596 
metabolism,  589 
mode  of  infection  with,  592 
morphology,  584 
staining,  584 
virulence,  594 

Kolle's  method  of  estimat- 
ing, 594 

vital  resistance,  589 
Petruschky's,  676 
proteus  vulgaris,  369 
cultivation,  369 
distribution,  369 
metabolic  products,  371 
morphology,  369 
pathogenesis,  371 
staining,  369 
pseudo-anthracis,  411 
pseudodiphtheria,  468,  470,  474 
chemistry,  475 
cultivation,  475 
differentiation    from    bacillus 

diphtheriae,  468,  474 
morphology,  475 
pathogenesis,  476 
staining,  475 
pseudodysentery,  699 
pseudoglanders,  784 
pseudo-influenza,  519 


854 


Index 


Bacillus,  pseudotetanus,  399 
pseudotuberculosis,  760 

cultivation,  760 

morphology,  760 

pathogenesis,  761 
psittacosis,  677 

cultivation,  677 

differentiation,  678 

isolation,  677 

metabolic  products,  677 

morphology,  677 

pathogenesis,  677 
pyocyaneus,  364 

cultivation,  365 

distribution,  364 

immunity  against,  368 

isolation,  365    • 

metabolic  products,  366 

morphology,  365 

pathogenesis,  367 

staining,  365 
pyogenes  foetidus,  666 
rhinoscleromatis,  785 

general  characteristics,  785 

pathogenesis,  786 
Sanarelli's,  682 
septicus  sputigenus,  493 
smegmatis,  757 

cultivation,  757 

Moeller's  method,  758 
Novy's  method,  757 

in  urine,  718 

morphology,  757 

pathogenesis,  758 

staining,  757 
suipestifer,  678 

agglutination,  68 1 

cultivation,  679 

metabolic  products,  680 

morphology,  679 

pathogenesis,  68 1 

toxin  of,  680 

vital  resistance,  680 
suisepticus,  60 1 

cultivation,  602 

general  characteristics,  60 1 

lesions  from,  603 

morphology,  60 1 

pathogenesis,  602 

staining,  602 

vital  resistance,  602 
tetani,  385 

antitoxin  against,  397 

bacilli  resembling,  399 

cultivation,  386 

Park's  method,  388 

distribution,  385 

immunity  against,  396 

isolation,  386 


Bacillus  tetani,  metabolic  products, 

390 

morphology,  386 
staining,  386 
toxic  products,  390 
vital  resistance,  389 
tuberculosis,  710 
agglutination,  745 
appearance  of  cultures,  726 
avium,  754 

cultivation,  755 
morphologic   peculiarities, 

755 

pathogenesis,  755 
staining,  755 
thermic  sensitivity,  755 
bacilli  resembling,  756 
bovis,  748 

lesions  produced  by,  749 
metabolic  products,  749 
morphology,  748 
pathogensis,  749 
staining,  749 
vegetation,  749 
channels  of  infection  for,  727 
gastro-intestinal  tract,  728 
placenta,  727 
respiratory  tract,  728 
sexual  apparatus,   729 
wounds,  730 
chemistry,  736 
cultivation,  722 

Koch's  method,  721 
distribution,  711 
effect  of  light  on,  727 
general  characteristics,  710 
in  feces,  staining,  718 
in  sections  of  tissue,  Ehrlich's 
method  of  staining,  719 
Gram's  method  of  stain- 
ing, 719 
Unna's  method  of  stain? 

ing,  719 

in  sputum,  staining,  714 
in  urine,  staining,  718 
isolation,  720 

antiformin  for,  720 
Dorset's  method,  723 
Frugoni's  method,  725 
Smith's  (T.)  method,  723 
morphology,  712 
pathogenesis,  727 
reaction,  727 
relation  to  oxygen,  727 
staining,  713 

Ehrlich-Koch  method,  715 
Ehrlich's  method,  713,   715 

for  sections,  719 
Gabbet's  method,  716 


Index 


855 


Bacillus      tuberculosis,      staining, 
Gram's  method,  for  sec- 
tions, 719 
in  feces,  718 
in  sputum,  714 
in  urine,  718 
Koch  method,  713 
Pappenheim's  method,   717 
Unna's  method,  for  sections, 

719 

Ziehl's  method,  716 
temperature  sensitivity,   727 
toxic  products,  737 
virulence,  735 
typhi  murium,  684 
cultivation,  684 
isolation,  684 
morphology,  684 
pathogenesis,  685 
staining,  684 
typhosus,  632 

bacilli  resembling,  663 

meat-poisoning  group,  665 
pneumonic  group,  665 
psittacosis  group,  665 
table    for    differentiation, 

664 

typhoidal  group,  665 
Buxton    and    Coleman's    me- 
dium for,  66 1 
Capaldi's  medium  for  plating, 

659 

colon  bacillus  and,  differentia- 
tion, 651 
cultural,  652 
serum,  651 
cultivation,  635 

Eisner's  method,  652 
Hiss'  method,  654 
.    Kashida's  method,  654 
Piorkowski's  method,  655 
Remy's  method,  653 
Rothberger's  method,  654 
distribution,  632 
effect    of    chemic   agents    on, 

637 

of  cold  on,  637 
general  characteristics,  632 
Hesse's   medium   for   plating, 

658 

in  blood,  643,  644 
in  feces,  isolation,  650 
in  gall-bladder,  640 
in  lower  animals,  644 
in  oysters,  298 
in  sputum,  644 
in  urine,  643 
invisible  growth,  636 
isolation,  634 


Bacillus  typhosus,  isolation,  Adami 
and  Chapin's  method, 
656 

Beckman's  method,  657 
Drigalski-Conradi    method, 

656 

Endo's  method,  659 
Petkowitsch's  method,  656 
Starkey's  method,  657 
Jackson's  culture-medium  for, 

66 1 

MacConkey's  medium  for,  66 1 
metabolic  products,  637 
mode  of  infection,  639 
morphology,  633 
pathogenesis,  639 
prophylactic  vaccination  with 

cultures  of,  646 
specific  therapy,  647 
staining,  633 

Ziehl's  method,  634 
thermal  death-point,  636 
toxic  products,  637 
vital  resistance,  636 
xerosis,  476 
chemistry,  477 
cultivation,  477 
morphology,  477 
pathogenesis,  477 
Y,  700 

zur  Nedden's,  450 
Bacteremia,  94 
Bacteria,  29 

anaerobic,    cultivation    of,    260. 
See    also    Anaerobic    bacteria, 
cultivation  of. 
associated   with    Bacillus   diph- 

theriae,  464 
with  suppuration,  341 
biology,  58 

Brownian  movement,  36 
capsule,  34 
cell-walls,  34 
Chester's  synopsis  of  groups  of, 

279 

chromogenic,  71 
classification,  29,  32 
colonies  of,  246,  251 

in  Esmarch's  tubes,  Esmarch 
instrument     for     counting, 

288 

types,  251 

colors  produced  by,  71 
cultivation,  224 
determination,  278 
distribution  of,  58 
fission  of,  36 
flagella  of,  35 
grouping  of,  32 


856 


Index 


Bacteria,     groups     of,     Chester's 
synopsis  of,  279 

higher,  41 

in  air,  58,  283 

quantitative     estimation,     by 

Hesse's  method,  284 
by  Petri's  method,  285 
by    Sedgwick's    method, 
286 

in  bladder,  85 

in  body,  80 

in  butter,  297 

in  conjunctiva,  82 

in  dental  caries,  82 

in  external  auditory  meatus,  82 

in  feces,  84,  85 

in  foods,  296 

in  ice,  289 

in  intestine,  83 

in  larynx,  85 

in  lungs,  85 

in  meat,  297 

in  milk,  296 

in  mouth,  82 

in  nose,  85 

in  oysters,  298 

in  sections  of  tissue,  method  of 
observing,  178 

in  shell-fish,  298 

in    skin   and    adjacent    mucous 
membranes,  80 

in  soil,  294 

Frankel's  method  of  estimat- 
ing number,  294 

in  stomach,  83 

in  trachea,  85 

in  urethra,  85 

in  uterus,  85 

in  vagina,  85 

in  water,  287 

method  of  determining  num- 
ber, 287 

Winslow  and  Willcomb's  direct 
method  of  enumeration  of, 
288 

influences  of  antibiosis  on  growth 

of,  63 

of  antiseptics  on  growth  of,  65 
of  association  on  growth  of,  63 
of  chemic  agents  on  growth  of, 

65 

of  electricity  on  growth  of,  62 
of  food  on  growth  of,  59 
of  light  on  growth  of,  61 
of  moisture  on  growth  of,  59 
of  movement  on  growth  of,  63 
of  oxygen  on  growth  of,  59 
of  reaction  on  growth  of,  61 
of  symbiosis  on  growth  of,  63 


Bacteria,  influences  of  temperature 

on  growth  of,  64 
of  x-rays  on  growth  of,  62,  63 

invasive  power,  89,  94 

isolation  of,  246 

Koch's  law  of  specificity,  23 

liquefaction  of  gelatin  by,  70 

living,  study  of,  172 

measurement  of,  200 

metabolism  of,  66 

methods  of  observing,  172 

morphology  of,  38 

motility,  35 

non-chromogenic,  71 

non-pathogenic,  76 

nucleus,  34 

of  plague  group,  597 

parasitic,  79 

pathogenic,  76,  79 

peptonization  of  milk  by,  76 

photographing,  200 

polar  granules,  34 

production  of  acids  by,  71 
of  alkalies  by,  71 
of  aromatics  by,  73 
of  disease  by,  76 
of  enzymes  by,  77 
of  fermentation  by,  67 
of  gases  by,  69 
of  nitrates  by,  74 
of  odors  by,  73 
of  phosphorescence  by,  73 
of  putrefaction  by,  68 

reproduction  of,  36 

saprophytic,  79 

size  of,  36 

specific,  339 

sporulation  of,  36 

staining,  174.     See  also  Staining. 

structure,  29,  34 

thermal  death-point  of,  deter- 
mination, 300 

thermophilic,  64 

toxic  power,  89 

transplantation  of,  from  culture- 
tube  to  culture-tube,  method 
of,  245 

virulence  of,  95.  See  also  Viru- 
lence. 

Bacterial  suspension  in  testing  op- 
sonic  value  of  blood,  308 
Bactericidal   strength   of   common 

disinfectants,  217 
Bacterination,  113 
Bacteriologic  syringes,  269 
Bacteriology,  evolution,  17 

of  air,  283 

of  foods,  296 

of  soil,  294 


Index 


857 


Bacteriology  of  water,  297 
Bacteriolysins,  166 
Bacteriolysis,  164,  166 
Bacterio-vaccination   in   staphylo- 

coccic  infections,  349 
Bacterium,  40 

coli  dysenteriae,  699 
pneumonias,  507 
termo,  369 
Bagdad  boil,  572 
Bain  fixateur,  192 

reducteur  et  reinforcateur,  192 
sensibilisateur,   192 
Balantidium  coli,  704 

animal  inoculation  with,  707 
cultivation,  707 
habitat,  707 

lesions  produced  by,  707 
morphology,  705 
motility,  706 
pathogenesis,  707 
reproduction,  706 
staining,  706 
transmission,  708 
diarrhea,  704 
lesions  of,  707 
transmission  of,  708 
Barber's  itch,  827 
Beckman's    method    of    isolating 

typhoid  bacillus,  657 
Behring's  method  of  determining 
potency  of  diphtheria  serum,  157 
Bench,  glass,  248 
Bergell  and  Meyer's  typhoid  serum, 

649 

Berkefeld  filter,  208 
Bichlorid  of  mercury  as  germicide, 

212 

Bile-salt  agar-agar,  662 
Biologic  contributions,  17 
Biology  of  bacteria,  58 
Biondi-Heidenhain       method      of 

staining  protozoa  in  tissue,  199 
Biscra  boil,  572 

button,  572 
Black  death,  581 
fever,  566 
molds,  47 
plague,  581 

Bladder,  bacteria  in,  85 
Blastomyces  dermatitis,  818 
cultivation,  821 
lesions,  823 
pathogenesis,  823 
staining,  821 
transmission,  823 
Blastomycetes,  43 

dermatitis,  44 
Blastomycetic  dermatitis,  818 


Blastomycosis,  818 

specific  organism,  820 

transmission,  823 
Blenorrhea,  450 
Blood  agar-agar  as  culture-media, 

234 

Bacillus  typhosus  in,  643,  644 
method  of  securing,  from  animals, 

273. 

opsonic  value  of,  bacterial  sus- 
pension in  testing,  308 
serum  in  testing,  311 
washed  leukocytes  in  test- 
ing, 3 1 1 

phagocy  tic -power  of,  307 
streptococcus   in,    in   scarlatina, 

355 
Blood-corpuscles  for  Wassermann 

reaction,  322 
titration  of,  324 
preparation  of,  164 
Blood-culture  in  typhoid  fever,  650 
Blood-serum,  alkaline,  as  culture- 
media,  237 
as  culture-media,  234 
mixture,    Loffler's,     as    culture- 
media,  236 
for  cultivation   of    Bacillus 

diphtheria,  454 
therapy,  26 
Boil,  Aleppo,  572 
bagdad,  572 
biscra,  572 
Delhi,  572 
Bordet-Gengou  bacillus,  488 

and  influenza   bacillus,    differ- 
ences between,  491 
cultivation,  490 
isolation,  489 
metabolic  products,  490 
morphology,  489 
pathogenesis,  490 
staining,  489 
phenomenon,  170 
Botkin's    apparatus     for     making 

anaerobic  cultures,  262 
Botulism,  299 
Botulismus,  69 

Bouillon  as  culture-media,  227 
preparation  of,  from  fresh  meat, 

227 

from  meat  extract,  229 
sugar,  230 

Bouillon-filtrate,  Denys',  742 
Bovine  actinomycosis,  803 
tuberculosis,  748 

communicability  to  man,  750 
prophylaxis,  753 
tuberculin  test  for,  754 


858 


Index 


Bromatotoxismus,  298 

Bronchopneumonia,  512 

Broth,  nitrate,  74 

Brownian  movement  of  bacteria, 
36 

Buboes  in  plague,  583 

Bubonic    plague,    581.     See    also 
Plague. 

Buchner's  method  of  making  anae- 
robic cultures,  263 

Buerger's    medium    for    isolating 
diplococcus  pneumoniae,  495,  496 

Buret  for  titrating  media,  225 

Burri's  India  ink  method  of  identi- 
fying treponema  p'allidum,  792 

Buton  d'Orient,  572 

Butter  bacillus,  760 
bacteria  in,  297 

Butyric  fermentation,  68 

Buxton    and    Coleman's    culture- 
medium  for  typhoid  bacillus,  66 1 


CABOT'S  method  of  treatment  of 

hydrophobia,  419 
Calmette's     ophthalmo-tuberculin 

reaction,  741 
Canal-water    bacillus,    capsulated, 

508 

Canned  goods,  poisoning  from,  299 
Capaldi's  medium  for  plating  ty- 
phoid bacillus,  659 
Capillary  glass  tubes,  243,  244 
Capsulated    canal-water    bacillus, 

508 
Capsule  bacillus,  509 

of  bacteria,  34 
Capsules,  collodion,  276 
Carbolic  acid  as  disinfectant,  215 
Carbuncle,  malignant,  401 
Carcinoma    of    stomach,    Oppler- 

Boas  bacillus  in,  83 
Caries,  dental,  bacteria  in,  82 
Carrasquilla's  leprosy  serum,  774 
Carriers,  typhoid,  640 
Catarrhal  inflammation,  439 

pneumonia,  512 
Catgut,  sterilization  of,  210 
Claudius'  method,  211 
cumol  method,  211 
Celloidin  embedding,  179 
Cells,  giant-,  in  tuberculosis,  732 

lepra,  772 

specific  affinity  of,  for  toxins,  93 
Cell-walls  of  bacteria,  34 
Ceratophyllus  fasciatus,  593 
Cercomonas  intestinalis,  709 
Cerebrospinal  fever,  423 

meningitis,  423 


Cerebrospinal  meningitis,  diplococ- 
cus of,  423 

Chamberland  filter,  208 

Chancroid,  442 

Chantemesse's  ocular  typhoid  re- 
action for  diagnosis  of  typhoid 
fever,  650 

Chapin  and  Adami's  method  for 
isolation  of  typhoid  bacillus,  656. 

Charbon,  400 

Cheese-poisoning,  299 

Chemic  agents,  influence  on  growth 

of  bacteria,  65 
contributions,  20 

Chester's  synopsis  of  groups  of 
bacteria,  279 

Chicken-cholera,  598 

Chlamydophrys  stercorea,  690 

Chlorin  as  germicide,  214 

Cholera,  Asiatic,  604 

immunity  against,  616 
prophylaxis,  617 
rice-water  discharges  of,  613 
sanitation  in,  618 
specific  organism  of,  discovery, 

605 

chicken-,  598 
de  poule,  598 
hog-,  678 
nostras,  619 

Chromogenesis,  71 

Chromogenic  bacteria,  71 

Chromogens,  66 

Cilia  of  protozoa,  56 

Cladothrix,  42 

Claudius'  method  of  sterilization 
of  catgut,  211 

Clegg  and  Musgrave's  agar-agar 
for  cultivating  amebas,  692,  694 

Clegg's  method  of  cultivation  of 
lepra  bacillus,  767 

Clonic  convulsions  in  tetanus,  393 

Clostridium,  37 

Clothing,  etc.,  disinfection  of,  221 

Coagulins,  140 

Cobralysin,  120 

Cocci,  38 

Coccidioidal  granuloma,  819 

Coccus,  diagram  illustrating  mor- 
phology, 39 

Cold,  effect  of,  on  Bacillus  typho- 

sus,  637 
influence  on  growth  of  bacteria, 

65 
Coleman  and  Buxton's  medium  for 

typhoid  bacillus,  66 1 
Coley's  mixture,  358 
Collodion  capsules,  276 

sacs,  preparation  of,  276,  277 


Index 


859 


Colonies,  246,  251 

in  Esmarch's  tubes,  Esmarch  in- 
strument for  counting,  288 

types  of,  251 

Colors  produced  by  bacteria,  71 
Comma  bacillus,  606 
Complement,    120,   141,    142,   144, 

145 
deviation  of,  phenomenon  result 

of,  167 
fixation,  170 
for  Wassermann  reaction,  321 

titration  of,  324 
Complicating  pneumonias,  513 
Concentrated  tuberculin,  739 
Congestive  chills  of  malaria,  539 
Conjunctiva,  bacteria  in,  82 
Conjunctival  reaction   in  typhoid 

fever,  650 
Conjunctivitis,   acute    contagious, 

446 

miscellaneous  organisms  in,  450 
Conorhinus  megistus,  565 
Conradi-Drigalski  method  of  iso- 
lation of  typhoid  bacillus,  656 
Contagion     from    Bacillus     diph- 

theriae,  468 
Contagious    conjunctivitis,    acute, 

446 

Contractile  vacuoles,  54 
Convulsions,  clonic,  in  tetanus,  393 

tonic,  in  tetanus,  393 
Coplin's  staining  jar,  181 
Copper  sulfate  as  germicide,  213 
Corks,  sterilization  of,  204 
Cornil  and  Babes'  method  of  stain- 
ing spirillum   cholerae  Asiaticee, 
608 
Corpuscles,    blood-,    for    Wasser- 

mann  reaction,  322 
titration  of,  324 
preparation  of,  164 
Cover-glass  forceps,  177 

preparations  for  general  exami- 
nation, 175,  176 
for  staining  protozoa,  196 
Gram's  method,  185 
Creolin,  215 
Croupous  pneumonia,  492 

lesions,  501 
Crude  tubercles,  733 

tuberculin,  738 
Cryptobia  borreli,  556 
Ctenopsylla  musculi,  593 
Culex  pipiens,  542 
Culicidae,  classification,  542 
Culture-media,  224 
agar-agar,  232 
alkaline  blood-serum,  237 


Culture  -  media,   blood  -  agar  -  agar, 

234 

-  blood-serum,  234 
bouillon,  227 

Deycke's  alkali-albuminate,  237 
Dunham's  solution,  240 
glycerin  agar-agar,  234 
litmus  milk,  239 
Loffler's    blood-serum    mixture, 

236 

milk,  239 

peptone  solution,  240 
Petruschky's  whey,  240 
potatoes,  237 
potato-juice,  238 
standard  reaction,  227 
sterilization  and  protection,  205 
Cultures,  242 
agar-agar,  255 
anaerobic,    Botkin's     apparatus 

for  making,  262 
Buchner's  method  of  making, 

263 
Frankel's  method  of  making, 

261 

Hesse's  method  of  making,  265 
Koch's  method  of  making,  266 
Liborius'  tube  for,  261 
Nichols  and  Schmitter's  meth- 
od of  making,  263 
Novy's  jars  for,  261 
Salomonsen's  method  of  mak- 
ing, 266 
Wright's   method   of   making, 

264,  267 
Zinsser's   method   of   making, 

264 
freshly    isolated,    standardizing, 

259 

gelatin  culture,  254 
in  fluid  media,  256 
manipulation,  technic,  243 
microscopic  study,  259 
plate,  242,  246 

apparatus  for,  247 
Esmarch's  tubes  for  making, 

250 

method  of,  247 
Petri's  dishes  for  making,  249 
pure,  242,  252 

special    methods   of   securing, 

256 

shake,  265 
standardizing    freshly    isolated, 

259 

study  of,  242 
upon  potato,  256 

Cumol  method  of  sterilization  of 
catgut,  211 


86o 


Index 


Cup,  pasteboard,  for  receiving  in- 
fectious sputum,  220 

Cutituberculin  reaction,  741 

Cytase,  144 

Cytolysins,  164 

Cytolysis,  164 

Cytoplasm,  34 
of  protozoa,  53 

Cytotoxins,  163 

Czenynke's   stain  for  Bacillus   in- 
fluenzae,  514 


DAVIS'    method   of   cultivation   of 

Ducrey's  bacillus,  443 
Death,  black,  581 
Death-point,  thermal,  of  bacteria, 

determination  of,  300 
Defensive  proteids,  128 
Dejecta,  disinfection  of,  220 
Delhi  boil,  572 
Dematium  albicans,  485 
Denecke,  spirillum  of,  622 
Dental  caries,  bacteria  in,  82 
Deny's  tuberculin,  742 
Dermatitis,  blastomycetic,  818 
Dermatomycosis,  824 
Dermatotuberculin  reaction,  741 
Desmon,  144 

Deviation  of  complement,  phenom- 
enon result  of,  167 
Deycke's  alkali-albuminate  as  cul- 
ture-media, 237 
Diarrhea,  balantidium,  704 
lesions  of,  707 
transmission  of,  708 
Digestive      apparatus,      infection 

through,  86 

Diluted  tuberculin,  739 
Diphtheria,  451 
antitoxin,  155,  47 1 
bleeding,  156 
effect  on  death-rate,  473 
immunization  of  animals,  156 
method  of  administration,  471 
paralysis  after  use  of,  472 
potency  of  serum,  157 
preparation  of  serum,  157 

of  toxin,  156 
prophylaxis,  471 
tetanus  after,  use  of,  473 
treatment  with,  471 
bacillus  of,  45 1 .    See  also  Bacillus 

diphtheria. 
bacteriologic  condition  of  throat 

in,  462 

contagion  from,  468 
diagnosis,  456 

bacteriologic,  456 


Diphtheria,  diagnosis,  outfit  for,  456 
lesions,  466 
mixed  infections,  466 
paralysis  after,  472 
pseudomembrane  of,  466 
relation   of   Bacillus   diphtherias 

to,  467 
of  streptococcus  pyogenes  to, 

.  354 

toxin,  460 

Diplobacillenconjunctivitis,  448 
Diplococcus,  38 

intracellularis  meningitidis,  423 
agglutination,  428 
cultivation,  426 
distribution,  424 
identification,  425 
isolation,  426 
metabolic  products,  428 
micrococcus  catarrhalis  and, 

differentiation,  425 
mode  of  infection  with,  430 
morphology,  424 
meningitidis,  pathogenesis,  428 
specific  therapy  with,  430 
staining,  425 
vital  resistance,  427 
of  Weichselbaum,  492 
pneumonias,  492 

animals  susceptible  to,  502 
bacteriologic  diagnosis,  503 
cultivation,  496 
distribution,  493 
general  characteristics,  492 
immune  serum  against,  505 
immunity  against,  505 
isolation,  495 
metabolic  products,  498 
morphology,  493 
pathogenesis,  498 
specificity  of,  502 
staining,  494 
toxic  products,  498 
virulence,  502 
vital  resistance,  497 
Diseases,  infectious,  339 

study  of,  21 
production  of,  76 
Dishes,  Petri's,  249 
Disinfectants,   common,   bacterici- 
dal strength,  217 
determination  of  value,  302 
inorganic,  213 
organic,  215 

Disinfection  and  sterilization,  201 
gaseous,  305 
of  air  of  sick-room,  2 1 1 
of  bodies  dead  of  infectious  dis- 
eases, 223 


Index 


86 1 


Disinfection  of  clothing,  etc.,  221 

of  dejecta,  211,  220 

of  furniture,  etc.,  222 

of  hands,  209 

of  instruments,  209 

of  ligatures,  209 

of  patient,  222 

of  sick-chambers,  211 

of  sutures,  209 

of  wound,  211 

use  of  sulphur  in,  218 

with  formaldehyd,  218 
Displacement  of  air  by  inert  gases 

in  anaerobic  cultures,  260 
Donovan-Leishman  body,  566 
Dorset's    method    of    isolation    of 

tubercle  bacillus,  723 
Dose,  amboceptor,  in  Wassermann 

reaction,  326 
Dourine,  562 

Drigalski-Conradi  method  of  iso- 
lation of  typhoid  bacillus,  656 
Drumstick,  37 
Ducrey's  bacillus,  442.       See  also 

Bacillus  of  Ducrey. 
Dumdum  fever,  566 
Dunham's  solution  as  culture-me- 
dium, 240 
Duval's  method  of  cultivation  of 

lepra  bacillus,  767,  768 
Dyscrasia,  101 
Dysentery,  687 

amebic,  689 

abscess  of  liver  in,  697 
lesions,  697 

bacillary,  699 
diagnosis,  704 
lesions,  702 
serum  therapy,  704 

bacillus  of,  699.    See  also  Bacillus 
dysentericB. 

endemics  of,  687 

epidemics  of,  687 


EBERTH-GAFFKY  bacillus,  632 .    See 

also  Bacillus  typhosus. 
Edema,  gaseous,  377 

bacillus  of,  377    See  also  Bacil- 
lus aerogenes  capsulatus. 
malignant,  374 

bacillus  of,  374.    See  also  Bacil- 
lus cedematis  maligni. 
Ehrlich-Koch   method   of  staining 

tubercle  bacillus,  715 
Ehrlich's    lateral-chain    theory    of 

immunity,  131 

method  of  determining  potency 
of  diphtheria  serum,  158 


Ehrlich's  method  of  staining  tuber- 
cle bacillus,  713,  715 
in  sections,  719 
solution,  182 
Electricity,  influence  on  growth  of 

bacteria,  62 
Electrozone,  214 
Elephantiasis  graecorum,  771 
Eisner's  method  of  cultivation  of 

typhoid  bacillus,  652 
Embedding,  119 
celloidin,  179 
glycerin-gelatin,  180 
paraffin,  180 
Emulsion,  bacillary,  745 
Encystment  of  protozoa,  57 
Endogenous  infections,  80 
Endomyces  albicans,  485 
Endo's  method  of  isolation  of  ty- 
phoid bacillus,  659 
!  Endospores,  37 
I  Endotheliolysis,  164 
l  Engle  and  Reichel's  stain  for  Negri 

bodies,  421 
Entamreba    buccalis    as    cause    of 

suppuration,  372 
coli,  691 

table  of  differential  features, 

693 
histologica,  689,  691 

morphology,  691 

reproduction,  692 

staining,  692 

table  of  differential  features, 

693 

lesions  produced  by,  697 
Mallory's  differential  stain  for, 

698 

metabolic  products,  695 
pathogenesis,  695 
tetragena,  690,  692 

isolation  and  cultivation,  692 

metabolic  products,  695 

pathogenesis,  695 

table  of  differential  features, 

693 

vital  resistance,  695 
vital  resistance,  695 
Enteric  fever,  632 
Enzymes,  production  of,  77 

tryptic,  71 
Eosin    and    methylene-blue    stain, 

1 86 
I  Epidemic  cerebrospinal  meningitis, 

423 

Epitheliolysins,  121 
I  Epitheliolysis,  164 
Ernst-Babes  granules,  34 
Erysipelas,  streptococcus  of,  361 


862 


Index 


Erythrasma,  824 

Esmarch's  instrument  for  counting 

colonies  of  bacteria  in  Esmarch 

tubes,  288 
tubes,  250 
Estivo-autumnal  fever,  parasite  of, 

537. 

Eurotium,  48 
Exhaustion   theory   of   immunity, 

125 

Exogenous  infections,  79 
Experimentation  upon  animals,  268 
Extracellular  toxins,  89,  91 

FACULTATIVE  anaerobes,  59 
Farcin  du  boeuf,  43 
Farcy,  781 
Farcy-buds,  781 
Faulnisszymoid,  24 
Favus,  828 

scutulum  formation,  828 

specific  organism  of,  829 
Febrile  tropical  splenomegaly,  566 
Feces,  Bacillus  typhosus  in,  isola- 
tion, 650 

bacteria  in,  84,  85 

spirillum  cholerae  Asiaticae  in, 
Schottelius'  method  of  detect- 
ing, 609 

tubercle  bacillus  in,  staining,  718 
Ferment,  putrefactive,  24 
Fermentation,  20,  67 

acetic,  67 

alcoholic,  67 

butyric,  68 

lactic  acid,  68 

Fermentation-tube,  Smith's,  69 
Filter,  Berkefeld's,  208 

Chamberland,  208 

Kitasato's,  208 

Pasteur-Chamberland,  207 

Petri's  sand,  for  air-examination, 
285 

Reichel's,  208 

Filtration,  sterilization  by,  208 
Finkler  and  Prior  spirillum,  619 
Fiocca's  method  of  staining  spores, 

189 

Fish  tuberculosis,  756 
Fishing,  252 
Fish-poisoning,  299 
Fission,  36 

results,  36 
Fixateur,  144,  145 
Fixation  of  complement,  170 
Fixed  virus  in  hydrophobia,  414 
Flagella,  35 

staining  of,  189.  See  also  Stain- 
ing flagella. 


Flagellates  in  intestines,  709 
Fleas,  plague  and,  592,  593 
Fleischner-giftung,  69 
Flexner  variety  of  dysentery  bacil- 
lus, 702 
Flexner's  antimeningococcus  serum, 

430 

Flies,  plague  and,  592 
Fluid  media,  cultures  in,  256 

Miiller's,  259 

Zenker's,  179 
Fluorescein,  366 
Fomites,  80 

foods  as,  296 
Food  as  fomites,  296 

bacteria  in,  296 

bacteriology  of,  296 

influence  on  growth  of  bacteria, 

59 

of  molds,  60 
of  protozoa,  60 
of  yeasts,  60 

poisons,  298 
Food-poisoning,  298 
Forceps,  cover-glass,  177 

Petri  dish,  249 

sterilization  of,  204 
Formaldehyd,  218 

as  germicide,  218 
Formalin,  215,  218 
Fowl  tuberculosis,  754 
Frambesia  tropica,  800 
Frankel's  instrument  for  obtaining 
earth  for  bacteriologic  study, 

295 
method  of  estimating  number  of 

bacteria  in  soil,  294 
of  making  anaerobic  cultures, 

261 
of  staining  diplococcus  intra- 

cellularis  meningitidis,  425 
Friedlander's  Bacillus  pneumoniae, 

507.     See  also  Pneumococcus. 
Frost's  plate  counter,  290 
Frothy  organs,  384 
Frugoni's   method   of   isolation   of 

tubercle  bacillus,  725 
Fungi,  ray-,  804 

Funnel  for  rilling  tubes  with  culture- 
media,  230 

Furniture,  etc.,  disinfection  of,  222 
Fusiform  bacillus,  478 


GABBET'S  method  of  staining  tuber- 
cle bacillus,  716 

GafTky-Eberth  bacillus,  632.  See 
also  Bacillus  typhosus. 

Galactotoxismus,  298 


Index 


863 


Gall-bladder,  Bacillus  typhosus  in, 

640 

Gamaleia,  spirillum  of,  625 
Gametocytes  of  plasmodium  falci- 

parum,  538 
malariae,  534 
vivax,  536 

Gartner's  bacillus,  675 
Gaseous  disinfection,  305 

edema,  377 

Gases,  production  of,  69 
Gastro-intestinal  tract  as  avenue  of 

infection    for    tubercle    bacillus, 

728 
Gelatin  as  culture-media,  231 

liquefaction  of,  70 

puncture  culture,  254 
Gelatin-media,  gelatin,  231 
Generation,  spontaneous,  doctrine 

of,  17 
Gengou-Bordet  bacillus,  488 

phenomenon,  170 
Genital         apparatus,        infection 

through,  88 

Germ  theory  of  disease,  23 
Germicidal     value     of     solutions, 

method  of  testing,  304 
Germicide,  201 

determination  of  value,  303 
Germination  of  spores,  38 
Ghoreyeb's     method     of     staining 

treponema,  789 
Giant-cells  in  tuberculosis,  731 
Gibson's  globulin  precipitation  for 

concentration      of       diphtheria 

serum,  159 
Glanders,  775 

bacillus  of,  775.    See  also  Bacillus 
mallei. 

diagnosis,  778 

in  human  beings,  783 

specific  organism,  775 
Glass  bench,  248 

tubes,  capillary,  243,  244 
Glassware  and   instruments,   ster- 
ilization of,  203 

protection  of,  203 
Globulin  precipitation  for  concen- 
tration of  diphtheria  serum,  159 
Glossina  morsitans,  561 

palpalis,  561,  563 

Glycerin     agar-agar     as     culture- 
media,  234 

Glycerin-gelatin,  embedding,  180 
Glycoproteids,  134 
Golden  staphylococcus,  343,  346 
Goldhorn's  method  of  staining  tre- 
ponema pallidum,  788 
Gonococcus,  431 


Gonorrhea,  431 

communication   of,    to   animals, 

436 

diagnosis,  435 
Gonotoxin,  435 

Gordon's  medium  for  differentia- 
tion   of    cholera    and    Kinkier- 
Prior  spirilla,  615 
Gram's  method  for  staining  tuber- 
cle bacillus  in  sections,  719 
of  staining,  182,  183,  184 
Nicolle's  modification,  185 
tubercle  bacillus  in  sections, 

719 
Gram-Weigert  method  of  staining, 

185 

Granulations  of  Schiiffner,  537 
Granules,  Babes-Ernst,  34 
metachromatic,  34 
polar,  34 

Granuloma,  coccidioidal,  819 
Grass  bacillus,  Moeller's,  758 


HAFFKINE'S  prophylactic,  596,  617 

Halogens  and  compounds  as  germi- 
cides, 214 

Hands,  disinfection  of,  209 

Hanging  block,  directions  for  pre- 
paring, 174 
drop,  173 

Hankin  and  Leumann's  method  for 
differential  diagnosis  of  plague 
bacillus,  588 

Haptophore  group,  131 

Hardening,  178 

Healed  tubercles,  736 

Heat,  influence  on  growth  of  bac- 
teria, 64 

Heidenhain-Biondi  method  of  stain- 
ing protozoa  in  tissue,  199 

Heidenhain's  method  of  staining 
protozoa  in  tissue,  199 

Heiman's  method  of  cultivation  of 
micrococcus  gonorrhoeas,  434 

Helcosoma  tropicum,  572,  573 

Hematozoa,  528 

Hemolysins,  163 

Hemolysis,  163 

Hemolytic  amboceptor  for  Wasser- 

mann  reaction,  323 
serum  for  Wassermann  reaction, 

titration  of,  324 
system  in  Wassermann  reaction, 

325 
Hemorrhagic  septicemia,  bacilli  of, 

597,  60 1 

Hemorrhagin,  162 
Herpes  circinatus,  824 


864 


Index 


Herpes  desquamans,  824 

tonsurans,  824 
Hesse's    apparatus    for    collecting 

bacteria  from  air,  284 
culture-medium  for  typhoid  ba- 
cillus, 658 

method  for  quantitative  estima- 
tion of  bacteria  in  air,  284 
of  making  anaerobic  cultures, 

265 

Higher  bacteria,  41 
Hill's  hanging  block,  174 
Hiss'    inulin-serum-water   test   for 
determining       pneumococcus, 
505 
method  of  cultivation  of  typhoid 

bacillus,  654 
of  staining  diplococcus   pneu- 

monise,  494,  495 
Hiss-Russell  variety  of  dysentery 

bacillus,  702 

Histoplasma  capsulatum,  573 
Histoplasmosis,  573 
Historical  introduction,  17 
Hoffmann's  bacillus,  474.    See  also 

Bacillus  pseudodiphtheria. 
Hog-cholera,  678 

Hogyes'  method  of  treating  hydro- 
phobia, 419 
Host,  78 

susceptibility  of,  101.      See  also 

Susceptibility. 
Hot-air  sterilizar,  203 
Hiihnercholera,  598 
Hydrogen  peroxid  as  germicide,  216 
Hydrophobia,  412 

communication  to  man,  412 

fixed  virus  in,  414 

scheme  for  intensive  treatment, 

418 

for  mild  treatment,  418 
street  virus  in,  414 
treatment,  414 

Cabot's  method,  419 
dilution  method,  419 
Hogyes'  method,  419 
Pasteur's    method,    114,    413, 

414,417 
Hypnococcus,  557 


ICE,  bacteria  in,  289 

Ice-cream  poisoning,  299 

Ichthyotoxismus,  299 

Immune  body,  120,  141,  142,  143, 

144,  145 
Immunity,  105 
acquired,  108 
accidentally,  109 


Immunity,  acquired,  active,  109 
experimentally,  109 
passive,  115 
through   accidental    infection, 

109 

through  bacterination,  113 
through  infection,  109 
through  inoculation,  109 
through  intoxication,  1 1 5 
through  vaccination,  no 
active,  106 

acquired,  109 
Ehrlich's    lateral-chain    theory, 

3i 

exhaustion  theory,  125 
explanation  of,  125 
Metschnikoff's  theory,  126 
natural,  107 
passive,  106 

acquired,  116 
relative,  106 
retention  theory,  125 
special  phenomena  of,  146 
synopsis  of  experimental  studies 

of,  118 

to  vaccination,  112 
variations  in,  112 
Incubating  oven,  258 
Incubator  for  opsonic  work,  315 
Index,  opsonic,  307,  316 
India   ink   method   of   identifying 

treponema  pallidum,  792 
Indol,  73 

Salkowski's  test  for,  73 
Infantile  kala-azar,  571 
Infection,  78 

avenues  of,  86,  98 

cardinal  conditions,  95 

endogenous,  80 

exogenous,  79 

experimental,  109 

mixed,  103 

proteus,  369 

sources  of,  79 

special  phenomena  of,  146 

sub-,  78 

terminal,  355 

through  digestive  apparatus,  86 

through  genital  apparatus,  88 

through  placenta,  88 

through   respiratory   apparatus, 

88 

through  skin,  86 
virulence  of,  95.     See  also  Viru- 
lence. 
Infectious  diseases,  339 

study,  21 

Inflammation,  catarrhal,  439 
Influenza,  514 


Index 


865 


Influenza,  diagnosis,  519 

Infusoria,  53 

Infusorial  life,  19 

Injection,  intra-abdominal,  271 

intrapleural,  271 

intravenous,  270 

into  rabbit,  method  of  making, 
270 

subcutaneous,  271 
Inoculation,  109 

advantages  of  vaccination  over, 
in 

animal,  271 

subcutaneous,  271 
Inorganic  disinfectants,  213 
Instruments  and  glassware,  steril- 
ization, 203 

disinfection  of,  209 

protection  of,  203 

surgical  sterilization  of,  211 
Intermediate  body,  120 
Intermittent  sterilization,  205 
Intestine,  bacteria  in,  83 

flagellates  in,  709 
Intoxication,    immunity    acquired 

by,  115 

Intra-abdominal  injection,  271 
Intracellular  toxins,  90 
Intrapleural  injections,  271 
Intravenous  injections,  270 

into  rabbit,  method  of  making, 

270 

Introduction,  historical,  17 
Invasive  power  of  bacteria,  89,  94 
Inulin-serum-water  test  of  Hiss  for 

determining  pneumococcus,  505 
lodin  terchlorid  as  germicide,  215 
Iron-hematoxylin  stain  for  proto- 
zoa, 199 

Isolation  of  bacteria,  246 
Itch,  barber's,  827 

JACKSON'S    medium    for    typhoid 

bacillus  in  water,  66 1 
Jactationstetanus,  394 
Jars,  Novy's,  for  anaerobic  cultures, 

261 

Javelle  water,  720 
Jaw,  lumpy,  803,  811 
Jennerian  vaccination,  no 

KALA-AZAR,  566 

diagnosis,  571 

infantile,  571 

lesions,  570 

transmission,  570 
Kashida's  method  of  cultivation  of 

typhoid  bacillus,  654 

55 


Kitasato's  filter,  207 
mouse-holder,  273 
Klatschpraparat,  256 
Klebs-Loffler  bacillus,  451.        See 

also  Bacillus  diphtheria. 
Knives,  sterilization  of,  204 
Koch-Ehrlich  method  of  staining 

tubercle  bacillus,  715 
Koch's  agglutination  test  for  tuber- 
cle bacillus,  746 

apparatus  for  coagulating  and 
sterilizing  blood-serum,  235, 
236 

law  of  specificity  of  bacteria,  23 
method  of  isolation  of  tubercle 

bacillus,  721 
of  making  anaerobic  cultures, 

266 
of   staining   tubercle   bacillus, 

7i3 

syringe,  269 
tuberculin,  739 

Koch-Weeks  bacillus,   446.        See 
also  Bacillus  of  Koch-Weeks. 

Kolle's    method    for    diagnosis    of 
plague,  594 

Krai's    method    of    cultivation    of 
Achorion  schonleinii,  831 

Kreotoxismus,  299 

Kiihne's  method  of  staining  Bacil- 
lus mallei,    777 


LA  FIEVRE  typhique,  632 
Lactic  acid  fermentation,  68 
Laitinen's  method  of  cultivation  of 

micrococcus  gonorrhoeas,  434 
Lamblia  intestinalis,  709 
Larynx,  bacteria  in,  85 
Latapie's  animal  holder,  272,  273 

instrument  for  preparing  tissue 

pulp,  165 

Latent  tuberculosis,  735 
Lateral  chain-theory  of  immunity, 

Ehrlich's,  131 

Law,  Koch's,  of  specificity  of  bac- 
teria, 23 

Leishman- Donovan  body,  566 
Leishmania  donovani,  566 
cultivation,  569 
distribution,  570 
morphology,  568 
transmission,  570 

furunculosa,  572 

infantum,  571 
Lepra  anaesthetica,  771,  773 

cells,  772 

nodosa,  771 
Leprolin,  774 


866 


Index 


Leprosy,  762 

anesthetic,  771,  773 
etiology,  763 
nodular,  771 
sanitation  in,  774 
serum,  774 
therapy  in,  774 
Leptothrix,  41 
Lethargy,  African,  554 
Leuconostoc,  39 
Leukocidin,  347 

Leukocytes,  washed,  in  testing  op- 
sonic  value  of  blood,  211 
Leumann  and  Hankin's  method  for 
differential   diagnosis   of   plague 
bacillus,  588 
Levaditi's  method  of  staining  tre- 

ponema  pallidum,  790 
Liborius'   tube  for  anaerobic  cul- 
tures, 261 
Life,  infusorial,  19 

spontaneous  generation  of,  doc- 
trine, 17 
Ligatures,  disinfection  of,  209 

sterilization  of,  210 
Light,  effect  of,  on  tubercle  bacillus, 

727 
influences  on  growth  of  bacteria, 

61 

Liquefaction  of  gelatin,  70 
Listerism,  25 
Litmus,  method  of  preparing,  239 

milk  as  culture-media,  239 
Litmus-lactose-agar-agar,  654 
Liver,  abscess  of,  in  amebic  dysen- 
tery, 697 

Lobar  pneumonia,  492 
Lockjaw,  385.     See  also  Tetanus. 
Loffler-Klebs  bacillus,  451.        See 

also  Bacillus  diphtheria. 
Loffler's    alkaline   methylene-blue, 
staining  Bacillus  diphtherias  with, 

452 

blood-serum  mixture  as  culture- 
media,  236 
method  for   detecting   spirillum 

cholerae  Asiaticse,  615 
of  cultivation  of  Bacillus  diph- 
therias, 454 

of  differentiating  typhoid  bacil- 
lus by  means  of  malachite 
green,  660 
of  staining,  182 

Bacillus  mallei,  776 
flagella,  189 
Luetin,  798 

Lugol's  solution,  dilute,  183 
Lumpy  jaw,  803,  811 
Lungs,  bacteria  in,  85 


Lupus,  99 
Lysin,  120 
Lysol,  215 
Ly ssa,  412.  See  also  Hydrophobia. 


MAcCoNKEY's    medium     for     ty- 
phoid bacillus,  66 1 

method  for  detecting  colon  bacil- 
lus in  water,  66 1 
Macfadyen's  typhoid  serum,  648 
Macrocytase,  127,  145 
Macrogametocyte,  531 
Macrophages,  126 
Madura-foot,  812 
Maladie  du  coit,  562 

du  sommeil,  554 
Malaria,  524 

ague-cake  of,  540 

algid  cases,  539 

and    mosquitoes,    relation,    526, 

54i 

congestive  chills  of,  539 
enlargement  of  spleen  in,  540 
estivo-autumnal  parasite  of,  537 
geographic  distribution,  524 
history,  524 
parasites  of,  525,  532 
animal  inoculation,  539 
cultivation,  539 
human,  532 

inoculation,  539 
pathogenesis,  539 
paroxysms  of,  524 
prophylaxis,  540 

human  beings,  540 
'    mosquitoes,  540 
quartan,  parasite  of,  532 
temperature  in,  524 
tertian,  parasite  of,  534 
Malignant  carbuncle,  401 
edema,  374 
polyadenitis,  581 
pustule,  407 
Mallein,  781 
Mallory's  differential  stain  for  en- 

tamoeba,  698 
method  of  staining,  186 
Malta  fever,  520 

bacteriologic  diagnosis,  521 
treatment,  522 
Mastigophora,  51 
Matino's  method  of  staining  proto- 
zoa, 198 
Measurement  of  micro-organisms, 

200 
Meat,  bacteria  in,  297 

extract,  preparation  of  bouillon 
from,  229 


Index 


867 


Meat,  fresh,  preparation  of  bouillon 

from,  227 
Meat-infusion,  228 
Meat-poisoning,  69,  299 
Meatus,    external    auditory,    bac- 
teria in,  82 
Medical  and  surgical  contributions 

to  history  of  bacteria,  21 
Mediterranean  fever,  520 
Megastomum  intestinalis,  709 
Meningitis,  cerebrospinal,  423 

diplococcus  of,  423 
Meningococcus,  423 
Mercuric  chlorid  as  germicide,  213 
Mercury,  bichlorid  of,  as  germicide, 

212 

Merismopedia,  38 
Merozoits,  529 
Metabolism  of  bacteria,  66 
Metachromatic  granules,  34 
Metschnikoff 's  theory  of  immunity, 

126 
Meyer  and  Bergell's  typhoid  serum, 

649 

Meyer's  syringe,  269 
Micrococcus,  38 
catarrhalis,  439 
cultivation,  440 
diplococcus  intracellularis  and, 

differentiation,  425 
morphology,  439 
pathogenesis,  441 
staining,  440 
gonorrhoeas,  431 
cultivation,  433 

Heiman's  method,  434 
Laitinen's  method,  434 
Wassermann's  method,  434 
Wertheim's  method,  433 
Wright's  method,  434 
Young's  method,  433 
diagnosis  of  gonorrhea  from, 

435 

distribution,  431 

immunization  against,  437 

isolation,  433 

morphology,  432 

pathogenesis,  436 

staining,  432 

toxic  products,  435 

vital  resistance,  434 
melitensis,  520 

cultivation,  520 

morphology,  520 

pathogenesis,  522 

staining,  520 

thermal  death-point,  520 
pasteuri,  26 
tetragenus,  361 


Micrococcus    tetragenus,    cultiva- 
tion, 362 
isolation,  362 
morphology,  361 
pathogenesis,  363 
staining,  362 
Microcytase,  127,  145 
Microgametocyte,  531 
Micromillimeter,  36 
Micro-organisms,    anaerobic,    cul- 
tivation of,  260.     See  also  An- 
aerobic bacteria,  cultivation  of. 
cultivation  of,  224 
in  air,  283 

measurement  of,  200 
methods  of  observing,  172 
of  plague  group,  597 
photographing,  200 
specific,  339 

structure  and  classification,  29 
Microphages,  126 
Microscopic  study  of  cultures,  259 
Microspira,  40 
Microsporon,  46 
Migula's  classification  of  bacteria, 

32 

Miliary  tubercle,  733 
Milk  as  culture-media,  239 
bacteria  in,  296 
litmus,  as  culture-media,  239 
peptonization  of,  76 
Milk-poisoning,  298 
Milzbrand,  400 
Mixed  infections,  103 

pneumonias,  513 
Mixture,  Coley's,  358 
Moeller's  grass  bacillus,  758 

method  of  cultivation  of  smegma 

bacillus,  758 
of  staining  spores,  188 
Moisture,  influence  on  growth  of 

bacteria,  59 
Molds,  46 
black,  47 
influence  of  food  on  growth  of,  60 

of  light  on  growth  of,  61 
Monilia  caudida,  485 
Morax-Axenfeld,  bacillus  of,  448 
cultivation,  449 
morphology,  449 
pathogenesis,  450 
staining,  449 

Morphology  of  bacteria,  38 
Morro's    method    of    diagnosis    of 

tuberculosis,  741 
Mosquitoes  and  malaria,  relation, 

526,  541 

and  yellow  fever,  577 
classification,  542 


868 


Index 


Mosquitoes,  destruction  of,  in  pre- 
vention of  malaria,  540 
Motility  of  bacteria,  35 
Mouse-holder,  272,  273 
Mouth,  bacteria  in,  82 
Movement,  influence  on  growth  of 
bacteria,  63 

of  protozoa,  55 
Mucor,  47 

conoides,  48 

corymbifer,  47,  48 

mucedo,  46,  47,  48 

mycosis,  48 

pusillus,  48 

ramosus,  47 

rhizopodiformis,  47 

septatus,  48 
Muguet,  484 

Muir  and  Ritchie's  method  of  stain- 
ing spores,  1 88 
Miiller's  fluid,  259 
Musgrave   and    Clegg's   agar-agar 

for  cultivation  of  amebas,  692, 

694 

Mussel-poisoning,  299 
Mycetoma,  812 

melanoid  form,  813,  816 

ochroid  variety,  813 

pale  variety,  813 
Mycoderma  vini,  485 
Mycophylaxis,  129 
Mycosis,  mucor,  48 
Mycosozins,  129 
Mytilotoxismus,  299 
Myzorrhynchus  psueudopictus,  542 


NAGANA,  560 

Needles,  platinum,  for  transferring 
bacteria,  243 

Negri  bodies,  421 

Neisser  and  Wechsberg's  phenome- 
non, 167,  168 

Neisser's  method  of  staining  Bacil- 
lus diphtherias,  453 

Nephelometer,  310 

Nephrolysins,  121 

Nephrotoxins,  121 

Nessler's  solution,  75 

Nichols  and  Schmitter's  method  of 
making  anaerobic  cultures,  263 

Nicolle's    modification    of    Gram's 
method  of  staining,  185 

Nitrate  broth,  74 

Nitrates,  formation  of,  74 
reduction  of,  74 

Nitrobacter,  74 

Nitrogen,  combination  of,  75 

Nitrosococcus,  74 


Nitroso-indol  reaction,  73 

Nitrosomonas,  74 

Nocard's  bacillus,  677 

Nodular  leprosy,  771 

Noguchi's    cutaneous    reaction    in 

diagnosis  of  syphilis,  798 
method  of  cultivation  of  trepo- 

nema  pallidum,  793 
modification  of  Wassermann  re- 
action, 335 

Non-chromogenic  bacteria,  71 
Non-malarial  remittent  fever,  566 
Non-pathogenic  bacteria,  76 
Nose,  bacteria  in,  85 
Novy's  jars  for  anaerobic  cultures, 

261 
method  of  cultivation  of  smegma 

bacillus,  757 
Nucleus  of  bacteria,  34 

of  protozoa,  55 

Nuttall  and  Welch's  method  of 
staining  bacillus  aerogenes  cap- 
sulatus,  380 


OBERMEIER'S  spirillum,  546 
Ocular  typhoid  reaction  of  Chante- 

messe,  650 

Odors,  production  of,  73 
Oidia,  44 

Oidium  albicans,  484 
cultivation,  486 
fermentation,  486 
immunity,  487 
metabolic  products,  486 
morphology,  485 
pathogenesis,  486 
Onychomycosis,  824 
Oocysts,  56,  531 
Ookinetes,  56,  531 
Ophidiomonas,  40 
Ophthalmia  neonatorum,  450 
Ophthalmo-tuberculin   reaction  of 

Calmette,  741 
of  Wolff-Eisner,  742 
Opisthotonos  in  tetanus,  393 
Oppler-Boas  bacillus  in  carcinoma 

of  stomach,  83 
Opsonic  index,  307,  316 

value  of  blood,  bacterial  suspen- 
sion in  testing,  308 
serum  in  testing,  311 
washed  leukocytes  in  test- 
ing, 311 

Opsonins,  127,  307 
Opsonizing  pipette,  313 
Optional  anaerobes,  59 
Organic  disinfectants,  215 
Oriental  sore,  572 


Index 


869 


Ornithodoros  moubata,  547,  551 
Oshida's  method  of  obtaining  rab- 
bit's cord  for  use  in  hydrophobia, 
414 

Oven,  incubating,  258 
Oxygen,    atmospheric,    absorption 
of,  in  anaerobic  cultures,  263 
exclusion  of,  in  anerobic  cul- 
tures, 265 
influence  on  growth  of  bacteria, 

59 

reduction  of,   in  anaerobic  cul- 
tures, 264 
relation  of  tubercle  bacillus  to, 

727 
Oysters,  bacteria  in,  298 


PALUDISM,  524 

Pappenheim's  method  of  staining 
tubercle  bacillus,  717 

Paracolon  bacilli,  665 

Paraffin  embedding,  180 

Paralysis  after  use  of   diphtheria 
antitoxin,  472 

Parasite,  78 
stomatitis,  484 

Parasites  of  malaria,  525,  532 
animal  inoculation,  539 
cultivation,  539 
human,  532 

inoculation,  539 
pathogenesis,  539 

Parasitic  bacteria,  79 

Paratyphoid  bacilli,  665 

Pariette's  culture  fluid,  657 

Park's  method  of  cultivating  teta- 
nus bacillus,  388 

Paroxysms  of  malaria,  524 

Pasteboard   cup   for   receiving   in- 
fectious sputum,  220 

Pasteur- Chamberland  filter,  207 

Pasteurian  vaccination,  113 

Pasteurization,  206 

Pasteur's     protective     inoculation 

against  anthrax,  409 
treatment  of  hydrophobia,   114, 
413,  414,  417 

Pathogenesis,  89 

Pathogenic  bacteria,  76,  79 
protozoa,  classification,  51 

Pathogens,  66 

Patient,  disinfection  of,  222 

Penicillium,  50 
crustaceum,  50 
glaucum,  50 
minimum,  50 

Peptone    solution    as    culture-me- 
dium, 240 


Peptonization  of  milk,  76 
Peroxid  of  hydrogen  as  germicide, 

216 

Pertussis,  488 
Pest,  581 
Petkowitsch's  method  of  isolation 

of  typhoid  bacillus,  656 
Petri  dish  forceps,  249 

dishes,  249 

method  for  quantitative  estima- 
tion of  bacteria  in  air,  285 

sand  filter  for  air-examination, 

285 
Petruschky's  bacillus,  676 

whey  as  culture-media,  240 
Pfeiffer's  method  of  staining,  181 

phenomenon,  120,  142,  166 
Phagocytes,  126 
Phagocytic  power  of  blood,  307 
Phagocytosis,  theory  of,  125 
Phagolysis,  126 
Phenomenon,  Bordet-Gengou,  170 

Neisser   and   Wechsberg's,    167, 
1 68 

Pfeiffer's,  120,  142,  166 

Theobald-Smith,  122 
Phlogosin,  347 

Phosphorescence,  production  of,  73 
Photogens,  66 
Photographing      micro-organisms, 

200 

Phylaxins,  128 
Pied  de  Madura,  812 
Pig  typhoid,  678 
Pink  eye,  446 
Piorkowski's  method  of  cultivation 

of  typhoid  bacillus,  655 
Pipette,  opsonizing,  313 
Pirquet's  method  of  cutaneous  di- 
agnosis of  tuberculo- 
^3,^740 
Lignieres'  modification, 

74i 
Pitfield's  method   of   staining  fla- 

gella,  191 

Smith's  modification,  191 
Pityriasis  versicolor,  824 
Placenta  as  avenue  of  infection  for 

tubercle  bacillus,  727 
infection  through,  88 
Plague,  581 
buboes  in,  583 
diagnosis,  593 
fleas  and,  592,  593 
flies  and,  592 

group  micro-organisms  of,  597 
history,  581 
immunity  against,  596 
mode  of  infection,  592 


87o 


Index 


Plague  pneumonia,  5 1 2 
sanitation  in.  595 
serum,  596,  597 
swine,  bacillus  of,  601 
Planococcus,  38 
Planosarcina,  39 
Plasmodium  falciparum,  529,  537 

gametocytes  of,  538 
malariae,  525,  529,  532 
gametocytes  of,  534 
spores  of,  429 
vivax,  529,  534 

developmental  cycle,  530 
gametocytes  of,  536 
Plasmolysis,  35 
Plate  cultures,  242,  246 
apparatus,  247 
Esmarch's  tubes  for  making, 

250 

method,  247 

Petri's  dishes  for  making,  249 
Platinum  needles  for  transferring 

bacteria,  243 
wires  for  bacteriologic  use,  243 

sterilization  of,  203 
Pneumobacillus,  507 
Pneumococcus,  492,  507 
cultivation,  509 
distribution,  508 
Hiss'  inulin-serum-water  test  for 

determining,  505 
infections   in   adults,    statistics, 

501,  502 

metabolic  products,  510 
morphology,  509 
pathogenesis,  510 
virulence,  512 
vital  resistance,  510 
Pneumonia,  492 
broncho-,  512 
catarrhal,  512 
complicating,  513 
croupous,  492 
lesions,  501 
lobar,  492 
mixed,  513 
plague,  512 
sanitation  in,  507 
tubercular,  512 
Pneumonias,  complicating,  513 

mixed,  513 

Pneumonic  plague,  512 
Poisoning,  food-,  298 
from  canned  goods,  299 
cheese-,  299 
fish-,  299 
ice-cream,  299 
meat-,  69,  299 
milk-,  298 


Poisoning,  mussel-,  299 

Poisons,  food,  298 

Polar  granules,  34 

Polyadenitis,  malignant,  581 

Polyceptor,  147 

Ponos,  571 

Postmortems,  275 

Potassium  permanganate  as  germi- 
cide, 214 

Potato  as  culture-media,  237 
cultures  upon,  256 
cutter,  Ravenel's,  238 

Potato-juice  as  culture-media,  238 

Precipitate,  specific,  146 

Precipitation,  globulin,  for  concen- 
tration  of   diphtheria   serum, 

159 

specific,  146 

Precipitins,  specific,  140 
Predisposition,  101 
Prescott  and  Winslow's  method  of 

preparing  litmus,  239 
Prior  and  Kinkier 's  spirillum,  619 
Prodigiosus  powder,  410 
Proteids,  defensive,  128 
Proteus  infection,  369 
Protista,  29 
Protozoa,  51 

cilia  of,  56 

classification,  51 

cytoplasm  of,  53 

encystment,  57 

influence  of  food  on  growth  of, 

60 
of  light  on  growth  of,  61 

living,  observation  of,  194 

movements  of,  55 

nucleus  of,  55 

pathogenic,  classification  of,  51 

reproduction  of,  56 

size  of,  56 

staining,  195.     See  also  Staining 
protozoa. 

structure,  53 
Pseudodiphtheria  bacillus,  468, 470, 

474.      See  also  Bacillus,  pseudo- 
diphtheria. 

Pseudodysentery  bacillus,  699 
Pseudoglanders  bacillus,  784 
Pseudo-influenza  bacillus,  519 
Pseudomonas,  40 
Pseudotetanus  bacillus,  399 
Pseudotuber.culosis,  760 
Psittacosis,  677 
Ptomains,  68 

definition  of,  68 
Pulex  cheopis,  593 

irritans,  593 
Pure  culture,  242,  252 


Index 


Pure  culture,  special   methods   of    Rossi's  method  of  staining  flagella, 

securing,  256  193 

Pustule,  malignant,  407 
Putrefaction,  20,  68 
Putrefactive  ferment,  24 
Pyemia,  94 
Pyocyanase,  77,  367 
Pyocyanin,  366 
Pyocyanolysin,  367 


QUARTAN  malarial  fever,  parasite 
of,  532 


RABBIT    septicemia,    bacillus    of, 

598,  601 

Rabies,  412.    See  also  Hydrophobia. 
Ravenel's  method  of  preparation  of 

agar-agar,  233 
potato  cutter,  238 
Ray-fungi,  804 
Receptors,  132,  153 
Refined  tuberculin,  739 
Regressive  schizogony,  532 
Reichel  and  Engle's  stain  for  Negri 

bodies,  421 
Reichel's  filter,  208 
Relapsing  fever,  546 

bacteriologic  diagnosis,  553 
course,  552 
immunity  to,  553 
lesions  of,  553 

Remittent  fever,  non-malarial,  56 
Remy's  method  of  cultivation  of 

typhoid  bacillus,  653 
Reproduction  of  bacteria,  36 

of  protozoa,  56 
Respiratory    apparatus,    infection 

through,  88 
tract  as  avenue  of  infection  for 

tubercle  bacillus,  728 
Retention  theory  of  immunity,  125 
Rhinoscleroma,  785 
Rhipicephalus  decoloratus,  546 
Rhizopoda,  51 
Rice-water    discharges    of    Asiatic 

cholera,  613 

Richardson's  method  of  differenti- 
ating typhoid  bacillus,  652 
Ringworm,  824 
of  body,  824 
of  scalp,  824 

Ritchie  and  Muir's  method  of  stain- 
ing spores,  1 88 

Romanowsky's  method  of  staining 
*  protozoa,  197 
Ross'  method  of  staining  protozoa, 


Rost's    method    of    cultivation    of 

lepra  bacillus,  766 
Rothberger's  method  of  cultivation 

of  typhoid  bacillus,  654 
Rouget  du  Pore,  26 
Roux's  syringe,  269 
R  tuberculin,  742 
Rubber  stoppers,   sterilization  of, 

204 


SACCHAROMYCES  hominis,  44,  818 

Saccharomycosis  hominis,  818 

Salkowski's  test  for  indol,  73 

Salomonsen's    method    of    making 
anaerobic  cultures,  266 

Salts  as  germicides,  213 

Sanarelli's  bacillus,  682 

Sand  filter,  Petri's,  for  air-examina- 
tion, 285 

Sapremia,  94 

Saprogens,  66 

Saprophytic  bacteria,  79 

Sarcina,  39 

Sarcoma,  Coley's  mixture  in,  358 

Scalp,  ringworm  of,  824 

Scarlatina,  streptococcus  in  blood 
in,  355 

Schaumorgane,  384 

Schizogony,  regressive,  532 

Schizont,  529 

Schlafkrankheit,  554 

Schmitter  and  Nichols'  method  of 
making  anaerobic  cultures,  263 

Schottelius'  method  of  detecting 
spirillum  cholerae  Asiaticae  in 
feces,  609 

of  securing  pure  cultures  of 
spirillum  cholerae  Asiaticae, 
609 

Schuffner's  granulations,  537 

Scissors,  sterilization  of,  204 

Scutulum,  828 

Sedgwick's  expanded  tube  for  air- 
examination,  285 
method  for  quantitative  estima- 
tion of  bacteria  in  air,  286 

Seitenkettentheorie,   131 

Semmelformig,  432 

Septicemia,  94 

hemorrhagic,  bacilli  of,  597 
rabbit,  bacillus  of,  598,  601 

Serum,  anticholera,  617 
antigonococcus,  437 
antimeningococcus,  430 
antipneumococcus,  505,  506 
antirabic,  422 


872 


Index 


Serum,  antistaphylococcus,  349 

antistreptococcus,  359 

antitubercle,  746 

antityphoid,  648 

anti  venomous,  161 

diagnosis  of  syphilis,  797 

disease,  123 

hemolytic,  for  Wassermann  re- 
action, titration  of,  324 

immune,  against  pneumococcus, 
505 

in  testing  opsonic  value  of  blood, 

3H 

leprosy,  774 
plague,  596,  597 
tetanus  antitoxic,  160 
therapy  in  anthrax,  410 
in  Asiatic  cholera,  617 
in  bacillary  dysentery,  704 
to  be  tested  in  Wassermann  re- 
action, 320 
Yersin's,  597 

Sexual  apparatus  as  avenue  of  in- 
fection for  tubercle  bacillus,  729 
Shake  culture,  265 
Sheep  corpuscles,  titration  of,  for 

Wassermann  reaction,  324 
Shell-fish,  bacteria  in,  298 
Shiga-Kruse  variety  of  dysentery 

bacillus,  702 
Shiga's  bacillus,   699.        See  also 

Bacillus  dysenteric. 
Siberian  pest,  400 
Sick-chambers,  disinfection  of,  211 
Sickness,  sleeping-,  554.      See  also 

Sleeping-sickness. 

Sick-room,  air  of,  disinfection,  211 
Silver  nitrate  as  germicide,  214 
Size  of  bacteria,  36 

of  protozoa,  56 

Skin  and  adjacent  mucous  mem- 
branes, bacteria  in,  80 
infection  through,  86 
Sleeping-sickness,  554 
prophylaxis,  563 
specific  organism,  555 
transmission,  559 

to  lower  animals,  562 
Smegma  bacillus,  757.       See  also 

Bacillus  smegmatis. 
Smith's  fermentation-tube,  69 
method  for  determining  bacillus 
coli   communis  in  water, 

291 

nature  of  gases,  70 
for  isolation  of  tubercle  bacil- 
lus, 723 

modification  of  Newman's  meth- 
od of  staining  flagella,  194 


Smith's  modification   of  Pitfield's 
method  of  staining  flagella,  191 
phenomenon,  122 
tube    for    isolation    of    tubercle 

bacillus,  723 
Soil,  bacteria  in,  294 

Frankel's  method  of  estimat- 
ing number,  294 
bacteriology  of,  294 
Soluble  toxins,  89,  91 
Solvent,  120 
Somers'  modification  of   Starkey's 

labyrinth,  658 
Soor,  484 
Sozins,  128 
Spasm  in  tetanus,  393 
Specific  micro-organisms,  339 
Spermatolysis,  164 
Spermatoxin,  121 
Spermotoxin,  121 

anti-,  121 

Spinal  cord  of  rabbit,  attenuation, 
for   use   in   hydrophobia, 
416 
method    of    obtaining,    for 

use  in  hydrophobia,  414 
Spirilla   resembling   cholera  spiril- 
lum, 619 
table    for    differentiating, 

631 
Spirillum,  38,  40 

cholerae     Asiaticae,     cultivation, 

609 
detection,  615 

Loffler's  method,  615 
distribution,  605 
general  characteristics,   604 
immunity  against,  616 
in  feces,  Schottelius'  method 

of  detecting,  609 
isolation,  608 

Loffler's  method  of  detect- 
ing, 615 

metabolic  products,  612 
morphology,  606 
pathogenesis,  613 
Schottelius'  method  of  mak- 
ing pure  cultures  of,  609 
serum  therapy  of,  617 
specificity,  615 
spirilla  resembling,  619 

table  for  differentiating, 
.    631 
staining,  608 

Cornil  and  Babes'  method, 

608 

toxic  products,  612 
vital  resistance,  611 
of  Denecke,  622 


Index 


873 


Spirillum  of  Denecke,  cultivation, 

623 

metabolic  products,  624 
morphology,  622 
pathogenesis,  624 
of  Kinkier  and  Prior,  619 
cultivation,  619 
metabolic  products,  622 
morphology,  619 
pathogenesis,  622 
staining,  619 
of  Gamaleia,  625 
cultivation,  625 
immunity  against,  628 
metabolic  products,  627 
morphology,  625 
pathogenesis,  627 
staining,  625 
vital  resistance,  627 
of  Obermeier,  546 
Spirochaeta,  40 
anserinum,  546 
carteri,  548 
dentinum,  546 
duttoni,  547,  548 
gallinarum,  546 
novyi,  548 

obermeieri,  546,  547,  548 
cultivation,  550 
general  characteristics,  548 
mode  of  infection,  550 
morphology,  548 
pathogenesis,  552 
staining,  550 
pallida,  787.    See  also  Treponema 

pallidum. 
pallidula,  801 
pertenuis,  80 1 
refringens,  787,  799 
theileri,  546 
vincenti,  479 

and     Bacillus    fusiformis,    re- 
lation, 479 
cultivation,  480 
morphology,  480 
pathogenesis,  483 
Spiromonas,  40 
Spirosoma,  40 
Spirulina,  40 
Spleen,  enlargement  of,  in  malaria, 

540 

Splenic  fever,  400 
Splenomegaly,  febrile  tropical,  566 
Spontaneous   generation,    doctrine 

of,  17 
Spores,  36 

germination  of,  38 
method  of  staining,  187.    See  also 
Staining  spores. 


Spores  of  plasmodium  malarise,  529 
Sporocysts,  56 
Sporozoa,  52 

furunculosa,  573 
Sporozoits,  528,  531 
Sporulation,  36 
Spotted  fever,  423 
Sputum,  Bacillus  typhosus  in,  644 
infectious,    pasteboard    cup    for 

receiving,  220 

tubercle  bacillus  in,  staining,  714 
Stain,    eosin   and   methylene-blue, 

1 86 
iron-hematoxylin,   for  protozoa, 

199 
Staining,  174 

aqueous  solution,  177 
flagella,  Loffler's  method,  189 
method  of,  189 
Pitfield's  method,  191 

Smith's  modification,  191 
Rossi's  method,  193 
Smith's  modification  of  New- 
man's method,  194 
Van  Ermengem's  method,  192 
Gram's  method,  182,  183,  184 

Nicolle's  modification,   185 
Gram-Weigert  method,  185 
jar,  Coplin's,  181 
Loffler's  method,  182 
Mallory's  method,  186 
Pfeiffer's  method,  181 
preparations  for  general  examin- 
ation, 975 
protozoa,  195 

Biondi-Heidenhain       method, 

199 

cover-glasses,  196 
Heidenhain's  method,  199 
in  tissue,  199 
Marino's  method,  198 
Romanowsky's  method,  197 
Ross'  method,  200 
slides,  196 

Wright's  method,  197 
simple  method,  176,  181 
spores,  Abbott's  method,  188 
Anjeszky's  method,  188 
Fiocca's  method,  189 
method  of,  187 
Moller's  method,  188 
Muir   and   Ritchie's   method, 

1 88 

stock  solutions,  177 
Zieler's  method,  186 
Standard  reaction  of  culture-media, 

227 

Standardizing  freshly  isolated  cul- 
tures, 259 


Index 


Staphylococci,    chief    types,    table 

of,  342 

Staphylococcus,  39 
citreus,  350 

epidermiditis  albus,  341 
golden,  343,  346 
pyogenes  albus,  343 
aureus,  343 
et  albus,  344 

agglutination,  349 
bacterio-vaccination,   349 
cultivation,  345 
distribution,  344 
isolation,  344 
morphology,  344 
pathogenesis,  347 
staining,  344 
thermal  death-point,  346 
toxic  products,  346 
treatment  with  serum,  349 
virulence,  349 
Staphylolysin,  347 
Starkey's    labyrinth    modified    by 

Somers,  658 
method  of  isolation  of  typhoid 

bacillus,  657 

Steam  sterilizer,  Arnold's,  205 
Stegomyia  calopus,  578 
fasciata,  578 

seu  calopus,  577 
Stemphylium  polymorpha,  485 
Sterilization  and  disinfection,  201 
and  protection  of  culture-media, 

205 

by  filtration,  208 
in  autoclave,  206 
intermittent,  205 
of  catgut,  210 

Cladius'  method,  211 
cumol  method,  211 
of  instruments  and  glassware,  203 
of  ligatures,  210 
of  platinum  wires,  203 
of  surgical  instruments,  211 
Sterilizer,  hot-air,  203 
Stewart's  cover-glass  forceps,  177 
Stock  solutions,  177 
Stomach,  bacteria  in,  83 

carcinoma  of,  Oppler-Boas  bacil- 
lus in,  83 

Stomatitis,  parasite,  484 
Street  virus  in  hydrophobia,  414 
Streptobacillus,  443 
Streptococcus,  39 
brevis,  351 
conglomeratus,  352 
diffusus,  352 
erysipelatis,  361 
in  blood  in  scarlatina,  355 


Streptococcus  longus,  361 
mucosus,  359 
pyogenes,  350 
cultivation,  351 
differential  features,  353 
isolation,  351 
morphology,  351 
pathogenesis,  354 
relation  to  diphtheria,  354 
staining,  351 
toxic  products,  357 
virulence,  356 
vital  resistance,  353 
viridans,  354 
Streptokolysin,  357 
Streptothrix,  42 
enteola,  42 
farcinica,  43 
Structure  of  bacteria,  34 

of  protozoa,  53 
Subcutaneous  injection,  271 

inoculation,  271 
Subinfection,  78 

Substance  sensibilisatrice,  144,  145 
Sucholotoxin,  68 1 
Sugar  bouillon,  230 
Suppuration,  339 
amebae  and,  372 
amoeba  kartulisi  as  cause,  372 

mortinatalium  as  cause,  373 
bacteria  associated  with,  341 
entamoeba  buccalis  as  cause,  372 
miscellaneous  organisms  of,  373 
Surgical  contributions  to  history  of 

bacteria,  21 

instruments,  sterilization  of,  211 
Susceptibility,  101 
diet  as  cause,  102 
exposure  to  cold  as  cause,  102 
fatigue  as  cause,  101 
inhalation  of  noxious  vapors  as 

cause,  1 01 

intoxication  as  cause,  102 
morbid  conditions  in  general  as 

cause,  103 

traumatic  injury  as  cause,  103 
Susotoxin,  680 
Sutures,  disinfection  of,  209 
Swine-plague,  bacillus  of,  60 1 
Symbiosis,  influence  on  growth  of 

bacteria,  63 
Synopsis,   proposed,   of  groups  of 

bacteria,  279 
Syphilis,  787 
bacillus  of,  787 
diagnosis,  797 
lesions  of,  797 

Noguchi's  cutaneous  reaction  in 
diagnosis  of,  798 


Index 


875 


Syphilis,  serum  diagnosis,  797 
Wassermann  reaction   for  diag- 
nosis of,  318.    See  also  Wasser- 
mann reaction. 
Syphilitic  antigen,  319 

titration  of,  328 
Syringe,  Altmann's,  269 
bacteriologic,  269 
Koch's,  269 
Meyer's,  269 
Roux's,  269 


TEMPERATURE  in  malaria,  524 
influence  on  growth  of  bacteria, 

64 

Terminal  infections,  355 
Tertian  malarial  fever,  parasite  of, 

^534. 
Tetamn,  391 

Tetanolysin,  91,  120,  131,  391,  394 
Tetanospasmin,  91,  391 
Tetanotoxin,  391 
Tetanus,  385 

after  use  of  diphtheria  antitoxin, 

4.73 
antitoxic  serum,  160 

antitoxin,  160,  397 

ascendens,  393 

bacillus,  385.      See  also  Bacillus 
tetani. 

clonic  convulsions  in,  393 

descendens,  393 

opisthotonos  in,  393 

pathogenesis,  394 

prophylactic  treatment,  398 

spasm  in,  393 

tonic  convulsions  in,  393 
Tetracoccus,  38 

Theobald-Smith  phenomenon,  122 
Theory,     Ehrlich's     lateral-chain, 
of  immunity,  131 

MetschnikofP s,  of  immunity,  126 
Thermal  death-point  of  bacteria, 

determination,  300 
Thermophilic  bacteria,  64 
Thrush,  484 
Tinea  circinata,  824 

favosa,  828 

imbricata,  824 

trichophytina,  824 

unguium,  824 

versicolor,  824 
Tongue,  wooden,  803,  811 
Tonic  convulsions  in  tetanus,  393 
Torrey's     antigonococcus     serum, 

437 

Toxemia,  94 
Toxic  power  of  bacteria,  89 


Toxins,  extracellular,  89 
intracellular,  89,  91 
soluble,  89,  91 
specific  action,  92 

affinity  of  cells  for,  93 
Toxophile  groups,  133 
Toxophore  group,  131 
Toxophylaxins,  129 
Toxosozins,  129 
T  R  tuberculin,  742 
Trachea,  bacteria  in,  85 
Treponema,  40 
pallidum,  787 

Burri's  India  ink  method  of 

identifying,  792 
cultivation,  793 

Noguchi's  method,  793 
distribution,  792 
general  characteristics,  787 
morphology,  788 
pathogenesis    and    specificity. 

795 
staining,  788 

Ghoreyeb's  method,  789 
Goldhorn's  method,  788 
Levaditi's  method,  790 
pertenue,  800 
cultivation,  802 
morphology,  801 
pathogenesis,  802 
staining,  80 1 

Trichomonas  intestinalis,  709 
Trichophyton,  46 
acuminatum,  825 
circonvulatum,  825 
crateriforme,  825 
effractum,  825 
exsiccatum,  825 
flavum,  825 
fulminatum,  825 
glabrum,  825 
megalosporon,  825 
microsporon,  825 
pilosum,  825 
plicatili,  825 
polygonum,  825 
regulare,  825 
sulphureum,  825 
tonsurans,  824 
cultivation,  825 
morphology,  825 
pathogenesis,  826 
umbilicatum,  825 
violaceum,  825 
Trikresol,  215 
Trommelschlager,  37 
Tropical  splenomegaly,  febrile,  566 

ulcer,  572 
Trypanosoma  avium,  556 


876 


Index 


Trypanosoma  brucei,  556 
castellani,  557 
cruzi,  564 

transmission,  565 
damoniae,  556 
equinum,  556 
equiperdum,  562 
gambiense,  554 
cultivation,  558 
morphology,  558 
pathogenesis,  562 
reproduction,  559 
staining,  558 
transmission,  559 

to  lower  animals,  562 
lewisi,  556. 
rajae,  556 
rotatorium,  556 
theileri,  556 
transvaliense,  556 
ugandense,  557 
various  species,  556 
Trypanosomiasis,  American,   564 
human,  554 
transmission,  565 
Tryptic  enzymes,  71 
Tse-tse-fly  disease,  560 
Tubercle  bacillus,  710.       See  also 

Bacillus  tuberculosis. 
Tubercles,  733 
crude,  733 
healed,  736 
miliary,  733 
of  Babes,  419 
Tubercular  abscess,  731 

pneumonia,  512 
Tuberculin,  738 
concentrated,  739 
crude,  738 
Denys',  742 
diluted,  729 
influence  on  tuberculous  tissue, 

740 

Koch's,  739 
preparation  of,  738 
refined,  739 

test  for  tuberculosis  of  cattle,  754 
Tuberculin-R,  742 
Tuberculin-T  R,  742 
Tuberculinic  acid,  737 
Tuberculocidin,  742,  743 
Tuberculosamin,  737 
Tuberculosis,  710 

bacillus  of ,  7 1  o.    See  also  Bacillus 

tuberculosis. 
bovine,  748 

communicability  to  man,  750 
prophylaxis,  753 
tuberculin  test  for,  754 


Tuberculosis,  diagnosis,  Calmette's 
ophthalmo-tuberculin  reac- 
tion, 741 

Morro's  method,  741 
von  Pirquet's  cutaneous  meth- 
od,- 740 
Lignieres'  modification, 

74i 

Wolff-Eisner      ophthalmo-tu- 
berculin method,  742 
fish,  756 
fowl,  754 
giant-cells  in,  731 
latent,  735 
lesions  of,  730 
of    cattle,    tuberculin    test    for, 

754 

prophylaxis,  747 
pseudo-,  760 
specific  organism,  711 
Tuberculous  abscess,  731 
Tubes,  capillary  glass,  243,  244 
Esmarch's,  250 

method  of  holding,  during  inocu- 
lation, 245 

Typhoid  carriers,  640 
fever,  632 

bacillus  of,  632 
bacteriologic  diagnosis,  649 
blood-culture  in,  650 
carriers,  640 

conjunctival  reaction  in,  650 
histologic  lesions,  642 
in  lower  animals,  644 
isolation  of  bacillus  from  feces 

in,  650 

pathogenesis,  639 
prophylactic    vaccination 

against,  646 
prophylaxis,  645 
specific  therapy,  647 
Widal  reaction  in,  649 
pig,  678 

reaction  of  Chantemesse,  650 
Typhus  abdominalis,  632 
Tyrotoxicon,  68,  299 
Tyrotoxismus,  299 


ULCER,  tropical,  572 

Umstimmung,  798 

Unna's  method  for  staining  tuber- 
cle bacillus  in  sections,  719 

Urethra,  bacteria  in,  85 

Urine,     Bacillus    tuberculosis     in, 

staining,  718 
Bacillus  typhosus  in,  643 
smegma  bacillus  in,  718 

Uterus,  bacteria  in,  85 


Index 


877 


VACCINATION,  no 
accidents  of,  112 
advantages  of,  over  inoculation, 

in 
bacterio-,   in   staphylococcic  in- 

fections, 349 
efficient,  112 
immunity  to,  112 
in  anthrax,  409 
inefficient,  112 
Jennerian,  no 
Pasteurian,  113 
prophylactic,     against     typhoid 

fever,  646 

Vaccine,  nature  of,  in 
Vaccines,  113 
Vaccinia,  112 
Vacuoles,  contractile,  54 
Vacuum,  formation  of,  in  anaerobic 

cultures,  260 
Vagina,  bacteria  in,  85 
Van  Brmengem's  method  of  stain- 

ing flagella,  192 
Vibrio,  40 
lineola,  799 
Metschnikovi,  625 
proteus,  619 
schuylkiliensis,  628 
cultivation,  628 
immunity  against,  629 
metabolic  products,  628 
morphology,  628 
pathogenesis,  629 
vital  resistance,  629 
tyrogenum,  622 
Vibrion  septique,  374 
Vibrionensepticaemia,  628 
Vincent's  angina,  478 
Virulence,  95 
decrease  of,  95 
increase  of,  96 

by  addition  of  animal  fluids 

to  culture-media,  97 
by  passage  through  animals,  96 
by  use  of  collodion  sacs,  96 
number  of  bacteria  influencing, 

97 
Virus,  fixed,  in  hydrophobia,  414 

street,  in  hydrophobia,  414 
von  Pirquet's  method  of  cutaneous 
diagnosis  of  tubercu- 
losis, 740 
Lignieres'  modification, 


WASHED  leukocytes  in  testing  op- 
sonic  value  of  blood,  311 

Wassermann  method  of  cultivation 
of  micrococcus  gonorrhoeae,  434 


Wassermann  reaction,  318 
amboceptor  dose  in,  326 

unit  in,  325,  326 
antigen  in,  319 

titration  of,  328 
blood-corpuscles  for,  322 

titration  of,  324 
complement  for,  321 

titration  of,  324 
hemolytic      amboceptor      for, 

323 
serum     for,     titration     of, 

324 

system  in,  325 
nature  of,  335 
Noguchi's  modification,  335 
reagents  employed,  318 
serum  to  be  tested,  320 
validity  of,  334 
Water,  Bacillus  coli  communis  in, 

291 
bacteria  in,  287 

method  of  determining  num- 
ber, 287 

Winslow  and  Willcomb's  di- 
rect method  of  enumeration 
of,  288 

bacteriology  of,  287 
colon  bacillus  in,  674 

MacConkey's    medium    for 

detecting,  66 1 

Wiirtz's  medium  for  detect- 
ing, 675 
typhoid    bacillus    in,    Starkey's 

method  of  isolating,  657 
Wechsberg  and  Neisser's  phenome- 
non, 167,  168 
Weeks-Koch  bacillus,   446.        See 

also  Bacillus  of  Koch-Weeks. 
Weigert-Gram  method  of  staining, 

185 

Welch    and   Nuttall's    method    of 
staining  bacillus  aerogenes  cap- 
sulatus,  379 
Welch's  method  of  staining  Dip- 

lococcus  pneumonias,  494 
Wertheim's  method  of  cultivation 

of  micrococcus  gonorrhoeae,  433 
Wesbrook's   types  of  Bacillus  dip- 

theriae,  452,  469 

Whey,    Petruschky's,    as    culture- 
medium,  240 
Whooping-cough,  488 
Widal  reaction,  149,  649 
Willcomb    and    Winslow's    direct 
method  of  enumeration  of  bac- 
teria in  water,  288 
Williams'  method  for  cultivation  of 
ameba,  695 


Index 


Winslow  and  Prescott's  method  of 

preparing  litmus,  239 
and   Willcomb's    direct   method 
of  enumeration  of  bacteria  in 
water,  288 
Wolff-Bisner     ophthalmo-tubercu- 

lin  reaction,  742 

Wolfhtigel's  apparatus  for  counting 
colonies  of  bacteria  upon  plates, 
287 
Wooden  apparatus,  sterilization  of, 

204 

tongue,  803,  811 
Wounds  as  avenue  of  infection  for 

tubercle  bacillus,  730 
disinfection  of,  211 
Wright's  method  of  cultivation  of 
micrococcus  gonorrhoeae,  434 
of    enumerating    bacteria    in 

fluid,  289 
of  making  anaerobic  cultures, 

264,  267 

of  staining  protozoa,  197 
Wiirtz's  method  for  detecting  colon 
bacillus  in  water,  675 


X-RAYS,    influence    on    growth    of 
bacteria,  62,  63 

YAWS,  800 

Y  bacillus,  700 

Yeasts,  43,  818 

influence  of  food  on  growth  of,  60 

of  light  on  growth  of,  61 
Yellow  fever,  576 

mosquitoes  and,  577 

prophylaxis,  580 
Yersin's  serum,  597 
Young's  method  of  cultivation  of 
micrococcus  gonorrhreae,  433 


fluid,  179 
Ziehl's  method  of  staining  Bacillus 

typhosus,  634 
tubercle  bacillus,  716 
Zieler's  method  of  staining,  186 
Zinsser's  method  of  making  anae- 

robic cultures,  264 
Zopf's  bacterium  pneumoniae,  507 
Zur  Nedden's  bacillus,  450 
Zygote,  531 


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DISEASES   Of   THE  EYE. 


Haab  and  DeSchweinitz's 
External  Diseases  qf  the  Eye 


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Atlas  and  Epitome  of  Ophthalmoscopy  and  Ophthalmoscopic 
Diagnosis.  By  Dr.  O.  HAAB,  of  Zurich.  Edited,  with  additions,  by 
G.  E.  DESCHWEINITZ,  M.  D.,  Professor  of  Ophthalmology,  University 
of  Pennsylvania.  With  152  colored  lithographic  illustrations  and  92 
pages  of  text.  Cloth,  $3.00  net.  In  Sannders'  Hand- Atlas  Series. 

THE  NEW   (2d)    EDITION 

The  great  value  of  Prof.  Haab's  Atlas  of  Ophthalmoscopy  and  Ophthalmo- 
scopic Diagnosis  has  been  fully  established  and  entirely  justified  an  English 
translation.  Not  only  is  the  student  made  acquainted  with  carefully  prepared 
Ophthalmoscopic  drawings  done  into  well-executed  lithographs  of  the  most  im- 
portant fundus  changes,  but,  in  many  instances,  plates  of  the  microscopic  lesions 
are  added.  The  whole  furnishes  a  manual  of  the  greatest  possible  service. 

The  Lancet,  London 

"We  recommend  it  as  a  work  that  should  be  in  the  ophthalmic  wards  or  in  the  library  of 
every  hospital  into  which  ophthalmic  cases  are  received." 


SAUNDERS*  BOOKS  ON 


Cradle's 
Nose,  Pharynx,  and  Ear 

Diseases  of  the  Nose,  Pharynx,  and  Ear.  By  HENRY  GRADLE, 
M.D.,  late  Professor  of  Ophthalmology  and  Otology,  Northwestern 
University  Medical  School,  Chicago.  Octavo  of  547  pages,  illustrated, 
including  two  full-page  plates  in  colors.  Cloth,  $3.50  net. 

INCLUDING  TOPOGRAPHIC  ANATOMY 

This  volume  presents  diseases  of  the  Nose,  Pharynx,  and  Ear  as  the  author 
has  seen  them  during  an  experience  of  nearly  twenty-five  years.  In  it  are 
answered  in  detail  those  questions  regarding  the  course  and  outcome  of  diseases 
which  cause  the  less  experienced  observer  the  most  anxiety  in  an  individual  case. 
Topographic  anatomy  has  been  accorded  liberal  space. 

Pennsylvania  Medical  Journal 

"This  is  the  most  practical  volume  on  the  nose,  pharynx,  and  ear  that  has  appeared 
recently.  ...  It  is  exactly  what  the  less  experienced  observer  needs,  as  it  avoids  the  confusion 
incident  to  a  categorical  statement  of  everybody's  opinion." 

Kyle's 
Diseases  of  Nose  and  Throat 


Diseases  of  the  Nose  and  Throat.  By  D.  BRADEN  KYLE,  M.  D., 
Professor  of  Laryngology  in  the  Jefferson  Medical  College,  Phila- 
delphia. Octavo,  797  pages;  with  219  illustrations,  26  in  colors. 
Cloth,  $4.00  net;  Half  Morocco,  $5.50  net. 

THE    NEW   (4th)    EDITION 

Four  large  editions  of  this  excellent  work  fully  testify  to  its  practical  value. 
In  this  edition  the  author  has  revised  the  text  thoroughly,  bringing  it  absolutely 
down  to  date.  With  the  practical  purpose  of  the  book  in  mind,  extended  con- 
sideration has  been  given  to  treatment,  each  disease  being  considered  in  full,  and 
definite  courses  being  laid  down  to  meet  special  conditions  and  symptoms. 
Pennsylvania  Medical  Journal 

"  Dr.  Kyle's  crisp,  terse  diction  has  enabled  the  inclusion  of  all  needful  nose  and  throat 
knowledge  in  this  book.  The  practical  man,  be  he  special  or  general,  will  not  search  in  vain 
for  anything  he  needs." 


GENITO-URINARY   AND    NOSE,     THROAT,     ETC.  9 

Greene  and  Brooks' 
Genito-Urinary  Diseases 

Diseases  of    the   Genito=Urinary  Organs  and  the  Kidney.      By 

ROBERT  H.  GREENE,  M.  D.,  Professor  of  Genito-Urinary  Surgery  at 
Fordham  University;  and  HARLOW  BROOKS,  M.  D.,  Assistant  Pro- 
fessor of  Clinical  Medicine,  University  and  Bellevue  Hospital  Medical 
School.  Octavo  of  639  pages,  illustrated.  Cloth,  $5.00  net;  Half 
Morocco,  $6.50  net. 

THE  NEW  (3d)  EDITION 

This  new  work  presents  both  the  medical  and  surgical  sides.  Designed  as  a 
work  of  quick  reference,  it  has  been  written  in  a  clear,  condensed  style,  so  that 
the  information  can  be  readily  grasped  and  retained.  Kidney  diseases  are  very 
elaborately  detailed. 

New  York  Medical  Journal 

"  As  a  whole  the  book  is  one  of  the  most  satisfactory  and  useful  works  on  genito-urinary 
diseases  now  extant,  and  will  undoubtedly  be  popular  among  practitioners  and  students." 

Gleason  on  Nose,  Throat, 
and  Ear 

A   Manual   of   Diseases  of   the    Nose,  Throat,  and    Ear.     By  E. 

BALDWIN  GLEASON,  M.  D.,  LL.  D.,  Clinical  Professor  of  Otology, 
Medico-Chirurgical  College,  Philadelphia.  I2mo  of  556  pages,  pro- 
fusely illustrated.  Flexible  leather,  $2.50  net. 

THE  NEW  (2d)  EDITION 

Methods  of  treatment  have  been  simplified  as  much  as  possible,  so  that  in 
most  instances  only  those  methods,  drugs,  and  operations  have  been  advised 
which  have  proved  beneficial.  A  valuable  feature  consists  of  the  collection  of 
formulas. 

American  Journal  of  the  Medical  Sciences 

"  For  the  practitioner  who  wishes  a  reliable  guide  in  laryngology  and  otology  there  are  few 
books  which  can  be  more  heartily  commended." 


American  Text-Book  of  Genito-Urinary  Diseases,  Syphilis,  and 
Diseases  of  the  Skin.  Edited  by  L.  BOLTON  BANGS,  M.  D.,  and 
W.  A.  HARDAWAY,  M.  D.  Octavo,  1229  pages,  300  engravings,  20 
colored  plates.  Cloth,  $7.00  net. 


io  SAUNDERS'   BOOKS    ON 

Goepp's 
Dental   State   Boards 

Dental  State  Board  Questions  and  Answers — By  R.  MAX  GOEPP, 
M.  D.,  author  "  Medical  State  Board  Questions  and  Answers."  Octavo 
of  428  pages.  Cloth,  $2.75  net. 

COMPLETE  AND  ACCURATE 

This  new  work  is  along  the  same  practical  lines  as  Dr.  Goepp's  successful  work 
on  Medical  State  Boards.  The  questions  included  have  been  gathered  from  reliable 
sources,  and  embrace  all  those  likely  to  be  asked  in  any  State  Board  examination 
in  any  State.  They  have  been  arranged  and  classified  in  a  way  that  makes  for  a 
rapid  resume  of  every  branch  of  dental  practice,  and  the  answers  are  couched  in 
language  unusually  explicit — concise,  definite,  accurate. 

The  practicing  dentist,  also,  will  find  here  a  work  of  great  value — a  work 
covering  the  entire  range  of  dentistry  and  extremely  well  adapted  for  quick 
reference. 

Haab  and  deSchweinitz's 
Operative  Ophthalmology 

Atlas  and   Epitome  of    Operative    Ophthalmology.       By  DR.  O. 

HAAB,  of  Zurich.  Edited,  with  additions,  by  G.  E.  DE  SCHWEINITZ, 
M.  D.,  Professor  of  Ophthalmology  in  the  University  of  Pennsylvania. 
With  30  colored  lithographic  plates,  1 54  text-cuts,  and  375  pages  of 
text.  In  Sounders'  Hand- Atlas  Series.  Cloth,  $3.50  net. 


Dr.  Haab's  Atlas  of  Operative  Ophthalmology  will  be  found  as  beautiful  and 
as  practical  as  his  two  former  atlases.  The  work  represents  the  author' s  thirty 
years'  experience  in  eye  work.  The  various  operative  interventions  are  described 
with  all  the  precision  and  clearness  that  such  an  experience  brings.  Recognizing 
the  fact  that  mere  verbal  descriptions  are  frequently  insufficient  to  give  a  clear 
idea  of  operative  procedures,  Dr.  Haab  has  taken  particular  care  to  illustrate 
plainly  the  different  parts  of  the  operations. 

Johns  Hopkins  Hospital  Bulletin 

"  The  descriptions  of  the  various  operations  are  so  clear  and  full  that  the  volume  can  well 
hold  place  with  more  pretentious  text-books." 


CHEMISTRY  AND 


Holland's  Medical 
Chemistry  and  Toxicology 

A  Text=Book  of  Medical  Chemistry  and  Toxicology.  By  JAMES 
W.  HOLLAND,  M.  D.,  Professor  of  Medical  Chemistry  and  Toxicology, 
and  Dean,  Jefferson  Medical  College,  Philadelphia.  Octavo  of  6j$ 
pages,  fully  illustrated.  Cloth,  $3.00  net. 

THE  NEW  (3d)  EDITION 

Dr.  Holland's  work  is  an  entirely  new  one,  and  is  based  on  his  forty  years' 
practical  experience  in  teaching  chemistry  and  medicine.  It  has  been  subjected  to 
a  thorough  revision,  and  enlarged  to  the  extent  of  some  sixty  pages.  The  additions 
to  be  specially  noted  are  those  relating  to  the  electronic  theory,  chemical  equilib- 
rium, Kjeldahl's  method  for  determining  nitrogen,  chemistry  of  foods  and  their 
changes  in  the  body,  synthesis  of  proteins,  and  the  latest  improvements  in  urinary 
tests.  More  space  is  given  to  toxicology  than  in  any  other  text-book  on  chemistry. 

American  Medicine 

"  Its  statements  are  clear  and  terse ;  its  illustrations  well  chosen;  its  development  logical, 
systematic,  and  comparatively  easy  to  follow.  .  .  .  We  heartily  commend  the  work." 

Ivy's  Applied  Anatomy  and 

Oral  Surgery  for  Dental  Students 


Applied   Anatomy  and   Oral   Surgery  for  Dental  Students.    By 

ROBERT  H.  IVY,  M.D.,  D.D.S.,  Assistant  Oral  Surgeon  to  the  Philadel- 
phia General  Hospital.  I2mo  of  280  pages,  illustrated.  Cloth,  $1.50 
net. 

FOR  DENTAL  STUDENTS 

This  work  is  just  what  dental  students  have  long  wanted— a  concise,  practical 
work  on  applied  anatomy  and  oral  surgery,  written  with  their  needs  solely  in 
mind.  No  one  could  be  better  fitted  for  this  task  than  Dr.  Ivy,  who  is  a  graduate 
in  both  dentistry  and  medicine.  Having  gone  through  the  dental  school,  he 
knows  precisely  the  dental  student's  needs  and  just  how  to  meet  them.  His 
medical  training  assures  you  that  his  anatomy  is  accurate  and  his  technic  modern. 
The  text  is  well  illustrated  with  pictures  that  you  will  find  extremely  helpful. 

H.  P.  Kuhn,  M.D.,  Western  Dental  College,  Kansas  City. 

"  I  am  delighted  with  this  compact  little  treatise.     It  seems  to  me  just  to  fill  the  bill." 


12  SAUNDERS*    BOOKS  ON 

Wells'  Chemical  Pathology 

Chemical  Pathology.  Being  a  discussion  of  General  Path- 
ology from  the  Standpoint  of  the  Chemical  Processes  Involved. 
By  H.  GIDEON  WELLS,  PH.  D.,  M.  D.,  Assistant  Professor  of 
Pathology  in  the  University  of  Chicago.  Octavo  of  549  pages. 
Cloth,  $3.25  net;  Half  Morocco,  $4.75  net. 

Wm.  H.  Welch,  M.  D.,  Professor  of  Pathology,  Johns  Hopkins  University. 

"  The  work  fills  a  real  need  in  the  English  literature  of  a  very  important  subject,  and 
I  shall  be  glad  to  recommend  it  to  my  students." 


The  New  (2d)  Edition 


Saxe's  Urinalysis 

Examination  of  the  Urine.  By  G.  A.  DE  SANTOS  SAXE,  M.  D., 
formerly  Instructor  in  Genito-Urinary  Surgery,  New  York  Post- 
graduate Medical  School  and  Hospital.  I2mo  of  448  pages,  fully 
illustrated.  Cloth,  $1.75  net. 

Francis  Carter  Wood,  M.  D.,    Adjunct  Professor  of  Clinical  Pathology,   Columbia   Uni- 
versity. 

"It  seems  to  me  to  be  one  of  the  best  of  the  smaller  works  on  this  subject  ;  it  is, 
indeed,  better  than  a  good  many  of  the  larger  ones." 

deSchweinitz  and  Randall   on  the  Eye,  Ear, 
Nose,  and  Throat 

American  Text-Book  of  Diseases  of  the  Eye,  Ear,  Nose,  and 
Throat,  Edited  by  G.  E.  DE  SCHWEINITZ,  M.D.,  and  B.  ALEX- 
ANDER RANDALL,  M.D.  Imperial  octavo,  1251  pages,  with  766 
illustrations,  59  of  them  in  colors.  Cloth,  $7.00  net;  Half  Mo- 
rocco, $8.50  net. 

Griinwald  and  Grayson  on  the  Larynx 

Atlas  and  Epitome  of  Diseases    of  the  Larynx.     By  Dr.  L. 

GRUNWALD,  of  Munich.  Edited,  with  additions,  by  CHARLES  P. 
GRAYSON,  M.D.,  University  of  Pennsylvania.  With  107  colored 
figures  on  44  plates,  25  text-cuts,  and  103  pages  of  text.  Cloth, 
$2.50  net.  In  Saunders1  Hand-Atlas  Scries. 


Mracek  and  Stelwagon's  Atlas  of  Skin 

Atlas  and  Epitome  of  Diseases  of  the  <Skin.  By  PROF.  DR. 
FRANZ  MRACEK,  of  Vienna.  Edited,  with  additions,  by  HENRY 
W.  STELWAGON,  M.D.,  Jefferson  Medical  College.  With  77  col- 
ored plates,  50  half-tone  illustrations,  and  280  pages  of  text.  In 
Saunders'  Hand-Atlas  Series.  Cloth,  $4.00  net. 


CHEMISTRY,   SKIN,  AND    VENEREAL    DISEASES.  13 

American  Pocket  Dictionary  New  (7th)  Edition 

THE  AMERICAN  POCKET  MEDICAL  DICTIONARY.  Edited  by  W.  A, 
NEWMAN  BORLAND,  M.  D.,  Editor  "  American  Illustrated  Medical 
Dictionary."  Containing  the  pronunciation  and  definition  of  the 
principal  words  used  in  medicine  and  kindred  sciences.  610  pages. 
Flexible  leather,  with  gold  edges,  $1.00  net;  with  thumb  index, 
#1.25  net. 
James  W.  Holland,  M.  D., 

Professor  of  Medical  Chemistry  and  Toxicology,  and  Dean,  Jefferson  Medical  College, 
Philadelphia, 

"  I  am  struck  at  once  with  admiration  at  the  compact  size  and  attractive  exterior.  ] 
can  recommend  it  to  our  students  without  reserve." 

Stelwagon's  Essentials  of  Skin  7th  Edition 

ESSENTIALS  OF  DISEASES  OF  THE  SKIN.  By  HENRY  W.  STEL- 
WAGON,  M.  D.,  PH.D.,  Professor  of  Dermatology  in  the  Jeffer- 
son Medical  College,  Philadelphia.  Post-octavo  of  291  pages, 
with  72  text-illustrations  and  8  plates.  Cloth,  $1.00  net.  In 
Saunders'  Question-  Compend  Series. 
The  Medical  News 

"  In  line  with  our  present  knowledge  of  diseases  of  the  skin.  .  .  .  Continues  to  main- 
tain the  high  standard  of  excellence  for  which  these  question  compends  have  been  noted." 

Wolffs  Medical  Chemistry  New  (7th)  Edition 

ESSENTIALS  OF  MEDICAL  CHEMISTRY,  ORGANIC  AND  INORGANIC. 
Containing  also  Questions  on  Medical  Physics,  Chemical  Physiol- 
ogy, Analytical  Processes,  Urinalysis,  and  Toxicology.  By  LAW- 
RENCE WOLFF,  M.  D.,  Late  Demonstrator  of  Chemistry,  Jefferson 
Medical  College.  Revised  by  A.  FERREE  WITMER,  PH.  G.,  M.  D., 
Formerly  Assistant  Demonstrator  of  Physiology,  University  of 
Pennsylvania.  Post-octavo  of  222  pages.  Cloth,  $1.00  net.  In 
Sounder^  Question- Compend  Series. 

Martin's  Minor  Surgery,  Bandaging,  and  the  Venereal 

Diseases  Second  Edition,  Revised 

ESSENTIALS  OF  MINOR  SURGERY,  BANDAGING,  AND  VENEREAL 
DISEASES.  By  EDWARD  MARTIN,  A.  M.,  M.  D.,  Professor  of  Clin- 
ical Surgery,  University  of  Pennsylvania,  etc.  Post-octavo,  166 
pages,  with  78  illustrations.  Cloth,  $1.00  net.  ///  Saunders* 
Question-  Compend  Series. 

Vecki's  Sexual  Impotence  New  (4th)  Edition-just  Ready 

SEXUAL  IMPOTENCE.  By  VICTOR  G.  VECKI,  M.  D.,  Consulting 
Genito-Urinary  Surgeon  to  Mt.  Zion  Hospital,  San  Francisco. 
I2mo  of  400  pages.  Cloth,  $2.25  net. 

Johns  Hopkins  Hospital  Bulletin 

"  A  scientific  treatise  upon  an  important  and  much  neglected  subject.  .  .  .  The 
treatment  of  impotence  in  general  and  of  sexual  neurasthenia  is  discriminating  and 
judicious." 


14  EYE,    EAR,    NOSE.    AND    THROAT. 

deSchweinitz    and    Holloway   on   Pulsating    Exoph- 
thalmos 

PULSATING  EXOPHTHALMOS.  An  analysis  of  sixty-nine  cases  not  pre- 
viously analyzed.  By  GEORGE  E.  DESCHWEINITZ,  M.  D.,  and  THOMAS 
B.  HOLLOWAY,  M.  D.  Octavo  of  125  pages.  Cloth,  $2.00  net. 

This  monograph  consists  of  an  analysis  of  sixty-nine  cases  of  this  affection 
not  previously  analyzed.  The  therapeutic  measures,  surgical  and  otherwise, 
which  have  been  employed  are  compared,  and  an  endeavor  has  been  made 
to  determine  from  these  analyses  which  procedures  seem  likely  to  prove  of 
the  greatest  value.  It  is  the  most  valuable  contribution  to  ophthalmic  liter- 
ature within  recent  years. 

British  Medical  Journal 

"The  book  deals  very  thoroughly  with  the  whole  subject  and  in  it  the  most  complete  account  of 
the  disease  will  be  found." 

Jackson  on  the  Eye  The  New  (2d)  Edition 

A  MANUAL  OF  THE  DIAGNOSIS  AND  TREATMENT  OF  DISEASES  OF  THE 
EYE.  By  EDWARD  JACKSON,  A.  M.,  M.  D.,  Professor  of  Ophthalmology, 
University  of  Colorado.  i2mo  volume  of  615  pages,  with  184  beautiful 
illustrations.  Cloth,  $2.50  net. 

The  Medical  Record,  New  York 

"  It  is  truly  an  admirable  work.  .  .  .  Written  in  a  clear,  concise  manner,  it  bears  evidence  of  the 
author's  comprehensive  grasp  of  the  subject.  The  term  '  multum  in  parvo '  is  an  appropriate  one  to 
apply  to  this  work." 

Grant  on   Face,   Mouth,   and  Jaws 

A  TEXT-BOOK  OF  THE  SURGICAL  PRINCIPLES  AND  SURGICAL  DISEASES 
OF  THE  FACE,  MOUTH,  AND  JAWS.  For  Dental  Students.  By  H.  HORACE 
GRANT,  A.  M.,  M.  D.,  Professor  of  Surgery  and  of  Clinical  Surgery, 
Hospital  College  of  Medicine,  Louisville.  Octavo  of  231  pages,  with 
68  illustrations.  Cloth,  $2.50  net. 

Preiswerk  and  Warren's  Dentistry 

ATLAS  AND  EPITOME  OF  DENTISTRY.  By  PROF.  G.  PREISWERK,  of 
Basil.  Edited,  with  additions,  by  GEORGE  W.  WARREN,  D.D.S.,  Pro- 
fessor of  Operative  Dentistry,  Pennsylvania  College  of  Dental  Surgery, 
Philadelphia.  With  44  lithographic  plates,  152  text-cuts,  and  343  pages 
of  text.  Cloth,  $3.50  net.  ///  Saunters'  Atlas  Series. 

Friedrich  and   Curtis  on  Nose,   Larynx,  and   Ear 

RHINOLOGY,  LARYNGOLOGy,  AND  OTOLOGY,  AND  THEIR  SIGNIFICANCE 
IN  GENERAL  MEDICINE.  By  DR.  E.  P.  FRIEDRICH,  of  Leipzig.  Edited 
by  H.  HOLBROOK  CURTIS,  M.  D.,  Consulting  Surgeon  to  the  New  York 
Nose  and  Throat  Hospital.  Octavo  volume  of  350  pages.  Cloth, 
$2.50  net. 


SAUNDERS'   BOOKS  ON 


Wolfs  Examination  of  Urine 

A  LABORATORY  HANDBOOK  OF  PHYSIOLOGIC  CHEMISTRY  AND 
URINE-EXAMINATION.  By  CHARLES  G.  L.  WOLF,  M.  D.,  Instructor  in 
Physiologic  Chemistry,  Cornell  University  Medical  College,  New 
York.  1 2mo  volume  of  204  pages,  fully  illustrated.  Cloth,  $1.25  net. 
British  Medical  Journal 

"  The  methods  of  examining  the  urine  are  very  fully  described,  and  there  are  at  the 
end  of  the  book  some  extensive  tables  drawn  up  to  assist  in  urinary  diagnosis." 

Jackson's  Essentials  of  Eye  Third  Revised  Edition 

ESSENTIALS  OF  REFRACTION  AND  OF  DISEASES  OF  THE  EYE.  By 
EDWARD  JACKSON,  A.  M.,  M.  D.,  Emeritus  Professor  of  Diseases  of 
the  Eye,  Philadelphia  Polyclinic.  Post-octavo  of  261  pages,  82  illus- 
trations. Cloth,  $1.00  net.  In  Saunders1  Question- Compend  Series. 
Johns  Hopkins  Hospital  Bulletin 

"  The  entire  ground  is  covered,  and  the  points  that  most  need  careful  elucidation 
are  made  clear  and  easy." 

Gleason's  Nose  and  Throat  Fourth  Edition,  Revised 

ESSENTIALS  OF  DISEASES  OF  THE  NOSE  AND  THROAT.  By  E.  B. 
GLEASON,  S.  B.,  M.  D.,  Clinical  Professor  of  Otology,  Medico- 
Chirurgical  College,  Philadelphia,  .etc.  Post-octavo,  241  pages,  1 12 
illustrations.  Cloth,  $1.00  net.  In  Saunders'  Question  Compends. 
The  Lancet,  London 

"  The  careful  description  which  is  given  of  the  various  procedures  would  be  sufficient 
to  enable  most  people  of  average  intelligence  and  of  slight  anatomical  knowledge  to 
make  a  very  good  attempt  at  laryngoscopy." 

Gleason's  Diseases  of  the  Ear  Third  Edition,  Revised 

ESSENTIALS  OF  DISEASES  OF  THE  EAR.     By  E.  B.  GLEASON,  S.  B., 
M.  D.,  Clinical  Professor  of  Otology,  Medico-Chirurgical  College, 
Phila.,  etc.     Post-octavo  volume  of  214  pages,  with   114  illustra- 
tions.    Cloth,  $  i. oo  net.     In  Saunders1  Question- Compend  Series. 
Bristol  Medico-Chirurgical  Journal 

"  We  know  of  no  other  small  work  on  ear  diseases  to  compare  with  this,  either  in 
freshness  of  style  or  completeness  of  information." 

Wilcox  on  Genito-Urinary  and  Venereal  Diseases 

The  New   (2d)   Edition 

ESSENTIALS  OF  GENITO-URINARY  AND  VENEREAL  DISEASES.  By 
STARLING  S.  WILCOX,  M.  D.,  Lecturer  on  Genito-Urinary  Diseases 
and  Syphilology,  Starling-Ohio  Medical  College,  Columbus.  I2mo 
of  321  pages,  illustrated.  Cloth,  $1.00  net.  Saunders'  Compends. 

Stevenson's  Photoscopy 

PHOTOSCOPY  (Skiascopy  or  Retinoscopy).  By  MARK  D.  STEV- 
ENSON, M.  D.,  Ophthalmic  Surgeon  to  the  Akron  City  Hospital. 
I2mo  of  126  pages,  illustrated.  Cloth,  $1.25  net. 

Edward  Jackson,  M.  D.,  University  of  Colorado. 

"  It  is  well  written  and  will  prove  a  valuable  help.  Your  treatment  of  the  emergent 
pencil  of  rays,  and  the  part  falling  on  the  examiner's  eye,  is  decidedly  better  than  any 
previous  account." 


